WO2006082414A1 - Detection et diagnostic du cancer - Google Patents

Detection et diagnostic du cancer Download PDF

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Publication number
WO2006082414A1
WO2006082414A1 PCT/GB2006/000364 GB2006000364W WO2006082414A1 WO 2006082414 A1 WO2006082414 A1 WO 2006082414A1 GB 2006000364 W GB2006000364 W GB 2006000364W WO 2006082414 A1 WO2006082414 A1 WO 2006082414A1
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boris
nucleic acid
polypeptide
sample
patient
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PCT/GB2006/000364
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English (en)
Inventor
Vivien D'arcy
France Docquier
Igor Chernukhin
Dawn Farrar
Elena Klenova
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Cancer Research Technology Limited
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Publication of WO2006082414A1 publication Critical patent/WO2006082414A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/4702—Regulators; Modulating activity
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING 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
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C12Q1/6886—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
    • 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
    • G01N33/57484—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumor, cancer, neoplasia, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides, metabolites
    • G01N33/57488—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumor, cancer, neoplasia, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides, metabolites involving compounds identifable in body fluids
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING 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
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/158—Expression markers
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/52—Predicting or monitoring the response to treatment, e.g. for selection of therapy based on assay results in personalised medicine; Prognosis

Definitions

  • the present invention relates to the detection of cancerous conditions or predisposition to cancerous conditions, and particularly, although not exclusively, relates to the detection of cancerous conditions or predisposition to cancerous conditions by detection of a BORIS polypeptide or nucleic acid, or a fragment of a BORIS polypeptide or nucleic acid .
  • Diagnosis of cancer at an early stage in cancer development is an important factor in improving the successful treatment of the cancer and in improving survival rates .
  • breast cancer is the most commonly diagnosed cancer after non-melanoma skin cancer, and is the second leading cause of cancer deaths after lung cancer .
  • breast cancer Because of its biological heterogeneity and wide spectrum of responsiveness to different treatments, breast cancer is a complex disease of difficult clinical management .
  • tumour markers proposed for the early detection and diagnosis of cancer are few in number and many suffer from disadvantages such as low expression levels making early detection difficult and unreliable .
  • For most clinical uses, including determination of susceptibility, screening, diagnosis and prognosis there are no known circulating tumour markers with established clinical utility. Indeed only a few markers have any established clinical utility, mostly for monitoring patients with detectable metastatic breast cancer .
  • cancer carcinoembryonic antigen
  • TPA tissue polypeptide antigen
  • GCDP gross cystic disease protein
  • PSA prostate specific antigen
  • MUC-I gene the products of the MUC-I gene .
  • Sensitivity levels for these markers has been found to be very low in early stage disease .
  • up to 20-30% of women with benign breast lesions have elevated levels of these markers, making the detection of new neoplastic tissue almost impossible .
  • markers are also related to epithelial malignancies other than breast cancer, and elevated levels may be a consequence of abnormalities and/or malignancies of the respiratory, gastrointestinal or reproductive " systems .
  • HER2/neu is a cellular transmembrane glycoprotein that, when overexpressed in breast cancer tissues, is associated with poor prognosis .
  • ECD extra-cellular domain
  • HER2/neu ECD extra-cellular domain
  • serum levels of HER2/neu ECD are elevated only in a sub-group of patients who overexpress HER2/neu at the tissue level . Therefore, a high false negative rate is associated with HER2/neu ECD as a marker for the diagnosis of breast cancer .
  • HER2/neu is overexpressed in other epithelial malignancies, including lung, ovarian, and gastrointestinal malignancies .
  • BORIS (Brother of the Regulator of Imprinted Sites ) is a CTCF (CCCTC binding factor) paralogue that is normally expressed only in the testis (usually in the cytoplasm of spermatocytes ) and is a member of the cancer-testis (C-T) gene family . Accordingly, BORIS is not normally expressed in women . BORIS has comparable exons, encoding an 11-zinc finger domain, to mammalian CTCF genes , but encodes N- and C- termini distinct from those in CTCF 1 . Human BORIS maps to 20ql3.2 and this chromosomal region has been shown to be amplified in many cancers 1 ' 2 .
  • CTCF normal functions include gene activation, repression / silencing, control of insulator function / imprinting, and also reading epigenetic marks .
  • CTCF and BORIS are expressed in a mutually exclusive pattern that correlates with re-setting of methylation marks during male germ cell differentiation 2 .
  • BORIS in normally BORIS-negative cells promotes cell growth, which can lead to transformation .
  • Abberantly expressed BORIS can take the place of CTCF in vivo and deregulate CTCF targets .
  • a rivalry caused by abnormal activation of BORIS in soma, especially in cells where it is never normally expressed, may lead to cancer development .
  • BORIS Owing to the normal expression of BORIS in testis only, BORIS is never normally active in females , but BORIS transcription is activated in varying proportions in a wide variety of cancers 2 . Notably, BORIS is expressed in some breast and ovarian tumours .
  • CT antigens are a category of tumour antigens with normal expression restricted to male germ cells in the testis but not in adult somatic tissues . In malignancy, this gene regulation is disrupted, resulting in CT antigen expression in a proportion of tumours of various types . Spontaneous humoral and cell-mediated immune responses "have been demonstrated against several CT antigens . Since CT antigens are immunogenic and highly restricted to tumours , their discovery has led directly to the development of antigen-specific cancer vaccines .
  • the inventors have detected BORIS in the granulocyte population of white blood cells taken from cancer patients .
  • Detection of a BORIS polypeptide or nucleic acid in the blood is proposed as a novel marker for following cancerous conditions .
  • the detection of BORIS in tissues other than the testis can be used as an indicator of the existence of a pathological condition, in particular of a cancerous condition .
  • the finding that BORIS polypeptide is aberrantly present in blood cells in cancer patients promotes BORIS as a specific circulating marker, the detection of which indicates the existence of a cancerous condition in the patient or of a predisposition of the patient to such a condition .
  • detection of blood-borne BORIS may be used in the diagnosis of a cancerous condition or in diagnosing a patient' s susceptibility to, or likelihood of, contracting a cancerous condition ( e . g . indicating a degree of risk that a patient may contract a cancerous condition) .
  • a blood borne marker of this kind is particularly advantageous in that the obtaining of a blood sample from a patient is a routine operation and avoids painful and sometimes difficult extraction of samples from specified tissues .
  • the blood sample may be readily processed, e . g . by centrifugation, to obtain a blood-derived sample being a specific sub-sample containing blood components of interest in which the presence of BORIS is to be measured .
  • the ease of obtaining blood samples and of testing also means that detection of BORIS may be used in formulating on-going prognosis indications in patients having an existing and previously diagnosed cancerous condition .
  • a reduction in the amount of BORIS may indicate a reduction in tumour malignancy and/or that a treatment is having a positive effect on tackling the cancerous condition .
  • detection of BORIS in blood may be used to assess the efficacy of a treatment or combination of treatments which the patient is receiving .
  • the detection of BORIS in the blood is an important aspect of the invention, the invention is not necessarily limited to expression of BORIS polypeptide or nucleic acid in blood components . Whilst not bound by any theory, two explanations for the presence of BORIS in blood components, particularly white blood cells, of cancer patients have been put forward by the inventors .
  • cellular components of the blood are aberrantly expressing BORIS nucleic acids which are translated into BORIS polypeptide which are expressed as a result .
  • This aberrant expression is abnormal and is a clear indicator of the existence of, predisposition to, or risk of contracting, a cancerous condition .
  • the inventors have noted the detection of BORIS polypeptide in the white blood cell population, particularly the granulocyte and peripheral blood mononuclear cell populations .
  • the presence of BORIS polypeptide in this cell population may be the result of phagocytosis of selected cells following the detection and destruction of unwanted proliferating cells by the patient' s immune system.
  • BORIS polypeptide expressed by those cells, may be taken up by the phagocytosing cell . Accordingly, it is possible that BORIS is not being expressed by the white blood cell population, rather it is being abnormally expressed in the unwanted proliferating cells forming all or part of the cancerous condition.
  • BORIS nucleic acid e . g . BORIS mRNA
  • BORIS is normally only expressed in the testis
  • detection of the presence of BORIS polypeptide or nucleic acid in any other tissue may form the basis of a method according to the present invention .
  • the detection of BORIS in blood of female patients may provide the basis for a particularly powerful and simple diagnostic test of the existence or predisposition to a cancerous condition in a female patient .
  • detection of BORIS in the blood may find particular use in the diagnosis and monitoring of breast, ovarian and uterine cancer .
  • CT antigens are a category of tumor antigens with normal expression restricted to male germ cells in the testis but not in adult somatic tissues . In malignancy, this gene regulation is disrupted, resulting in CT antigen expression in a proportion of tumors of various types . Spontaneous humoral and cell-mediated immune responses have been demonstrated against several CT antigens . Since CT antigens are immunogenic and highly restricted to tumors, their discovery has led directly to the development of antigen-specific cancer vaccines .
  • the present invention relates to the detection of a cancerous condition or predisposition to a cancerous condition .
  • the present invention may relate to a method comprising detecting the presence or absence of a BORIS polypeptide or a nucleic acid encoding a BORIS polypeptide, or a fragment of said polypeptide or nucleic acid.
  • a method of diagnosing a cancerous condition, or predisposition to a cancerous condition comprising detecting the presence of a BORIS polypeptide or a nucleic acid encoding a BORIS polypeptide, or a fragment of said polypeptide or nucleic acid .
  • the method preferably comprises detecting the presence of a BORIS polypeptide , nucleic acid or fragment thereof in a sample taken from a patient . Accordingly, the method may detect the expression of a BORIS polypeptide or nucleic acid (e . g . BORIS mRNA) in the sample .
  • the method may comprise detection, in selected cells, of gene amplification of the genomic nucleotide sequence encoding BORIS .
  • the gene amplification detected may be an abnormal gene amplification leading to aberrant expression of BORIS .
  • Detection of a BORIS polypeptide or a nucleic acid encoding a BORIS polypeptide, or a fragment of said polypeptide or nucleic acid, in non-testis tissue is indicative of the presence of, or predisposition to, a cancerous condition .
  • the method of diagnosis may be an in vitro method performed on the patient sample, or following processing of the patient sample . Once the sample is collected, the patient is not required to be present for the in vitro method of diagnosis to be performed and therefore the method may be one which is not practised on the human or animal body .
  • an in vitro method of diagnosing a cancerous condition in a patient comprising the step of detecting in a patient blood sample, or blood-derived sample, the presence of a BORIS polypeptide or a nucleic acid encoding a " BORIS polypeptide, or a fragment of said polypeptide or nucleic acid.
  • a method of assessing the effectiveness of treatment of a cancerous condition by analysing the presence of a BORIS polypeptide or a nucleic acid encoding a BORIS polypeptide , or a fragment of said polypeptide or nucleic acid.
  • a method of assessing the effectiveness of treatment of a cancerous condition in a patient comprising the step of analysing in a patient blood sample, or blood-derived sample, the presence of a BORIS polypeptide or a nucleic acid encoding a BORIS polypeptide, or a fragment of said polypeptide or nucleic acid.
  • the analysis of BORIS presence may comprise monitoring the presence or absence of a BORIS polypeptide or nucleic acid in a sample taken from a patient . This may involve monitoring the expression of a BORIS polypeptide or nucleic acid in the sample or in specific components of the sample .
  • the analysis may comprise measuring the amount of BORIS polypeptide or nucleic acid in the sample . This may comprise quantitative measurement of the amount of BORIS polypeptide or nucleic acid which is present or is expressed, e . g . using quantitative techniques such as quantitative western blot or quantitative northern blot . Alternatively the analysis may comprise a qualitative analysis , e . g . by monitoring the continued presence or absence of BORIS polypeptide or nucleic acid by microscopy.
  • the assessment of effectiveness of treatment may be made by monitoring the change in the presence of a BORIS polypeptide or nucleic acid, e . g . by monitoring a decrease or increase in BORIS polypeptide or nucleic acid presence or expression .
  • methods are provided for : (i) monitoring a cancerous condition in a patient; (ii ) predicting of a patient' s response to therapeutic treatment of a cancerous condition; (iii) assessing the prognosis of a patient having a cancerous condition; (iv) screening a patient for the presence of a cancerous condition; and/or (v) determining the susceptibility of a patient to developing or contracting a cancerous condition, wherein said method comprises detecting the presence of a BORIS polypeptide or a nucleic acid encoding a BORIS polypeptide , or a fragment of said polypeptide or nucleic acid in a sample taken from the patient . Detection of a BORIS polypeptide or nucleic acid is preferably in a patient blood sample, or blood-derived sample .
  • SEQ ID No .1 ( Figure HA) represents the nucleotide sequence corresponding to human BORIS and SEQ ID No .3 ( Figure HB) represents the nucleotide sequence corresponding to murine BORIS .
  • SEQ ID No .2 ( Figure 12A) represents the amino acid sequence corresponding to human BORIS polypeptide and SEQ ID No .4 ( Figure 12B) represents the amino acid sequence corresponding to murine BORIS polypeptide .
  • a BORIS nucleic acid or nucleic acid encoding a BORIS polypeptide may be any nucleic acid ( DNA or RNA) having a nucleotide sequence having a specified degree of sequence identity to one of SEQ ID No . s 1 or 3 or to the nucleotide sequence (comprising nucleotides 1 to 3493 ) set out in Figure 13 , an RNA transcript of any one of these sequences, a fragment of any one of the preceding sequences or the complementary sequence of any one of these sequences or fragments .
  • a BORIS nucleic acid may further relate to any nucleic acid encoding a BORIS polypeptide .
  • the specified degree of sequence identity may be from at least 60% to 100% sequence identity.
  • the specified degree of sequence identity- may be one of at least 65% , 70% , 75%, 80% , 85% , 86% , 87% , 88% , 89% , 90% , 91% , 92%, 93% , 94%, 95% , 96%, 97%, 98% or 99% identity .
  • a BORIS polypeptide may be any peptide, polypeptide or protein having an amino acid sequence having a specified degree of sequence identity to one SEQ ID No . s 2 or 4 or to the amino acid sequence set out in Figure 13 or to a fragment of one of these sequences .
  • the specified degree of sequence identity may be from at least 60% to 100% sequence identity .
  • the specified degree of sequence identity may be one of at least 65% , 70% , 75% , 80% , 85% , 86% , 87% , 88% , 89% , 90% , 91% , 92% , 93% , 94% , 95%, 96% , 97% , 98% or 99% identity .
  • a fragment may comprise a nucleotide or amino acid sequence encoding a portion of the corresponding full length sequence .
  • the corresponding full length sequence may be one of SEQ ID No . s 1 to 4 or one of the sequences set out in Figure 13.
  • Said portion may be of defined length and may have a defined minimum and/or maximum length .
  • the fragment may comprise at least, i . e . have a minimum length of, 10 , 11, 12 , 13 , 14 , 15 , 16, 17 , 18 , 19, 20, 25 , 30 , 40, 50 , 60, 70, 80 , 85, 90, 95, 96, 97, 98 or 99% of the corresponding full length sequence .
  • the fragment may have a maximum length, i . e . be no longer than, 10, 11 , 12 , 13 , 14 , 15 , 16, 17 , 18 , 19, 20, 25, 30, 40, 50 , 60 , 70, 80 ,- 85, 90, 95 , 96, 97 , 98 or 99% of the corresponding full length sequence .
  • the fragment may comprises at least, i . e . have a minimum length of, 10 nucleotides or amino acids , more preferably at least 15 , 16, 17, 18 , 19 , 20 , 21 , 22 , 23 , 24 , 25, 30, 40, 50 , 100 , 150 , 200 , 300, 400, 500, 600, 700, 800 , 900 , 1000 , 1100 , 1200 , 1300 , 1400 , 1500 , 1600, 1700 , 1800 , 1900 2000, 2100, 2200, 2300, 2400, 2500, 2600, 2700 , 2800 , 2900, 3000, 3100, 3200 , 3300 or 3400 nucleotides or amino acids .
  • the fragment may have a maximum length of, i . e . be no longer than, 10 nucleotides or amino acids , more preferably no longer than 15, 16, 17 , 18 , 19, 20, 21, 22 , 23 , 24 , 25, 30 , 40, 50, 100 , 150 , 200 , 300 , 400 , 500 , 600 , 700 , 800 , 900 , 1000, 1100, 1200 , 1300, 1400 , 1500, 1600, 1700 , 1800 , 1900 2000 , 2100 , 2200 , 2300 , 2400 , 2500, 2600, 2700, 2800 , 2900 , 3000 , 3100 , 3200, 3300 or 3400 nucleotides or amino acids .
  • the fragment length may be anywhere between the said minimum and maximum length .
  • detection of a BORIS polypeptide or nucleic acid may be performed by any appropriate means available to the person skilled in the art .
  • One way of detecting the presence of a BORIS polypeptide or nucleic acid is by using anti-BORIS antibodies which may be monoclonal or polyclonal antibodies . These may be used to detect the presence or expression of a BORIS polypeptide, or BORIS epitope forming part of a BORIS polypeptide by assays involving binding between a BORIS polypeptide or nucleic acid (or fragment thereof) and the anti-BORIS antibody . Suitable antibodies may be labelled with other compounds to aid visualisation, e . g . using fluorescent or radio- labels or secondary antibodies linked to similar labelling compounds . In one arrangement, detection of BORIS polypeptide may be by western blotting techniques using anti-BORIS antibodies .
  • Quantitative western blot using radio-labelled antibodies may be used to determine a relative amount of a BORIS polypeptide or nucleic acid in a sample .
  • nucleic acid probes may be constructed to comprise nucleic acid sequences predetermined to have a high degree of sequence complementarity with a selected portion of a BORIS nucleic acid such that the probe will hybridise with the BORIS nucleic acid under high or very high stringency conditions, preferably under very high stringency conditions .
  • High stringency conditions may be provided by controlling various parameters of the hybridisation reaction . These have been described previously (e . g. see Sambrook J, Russell DW . Molecular cloning : A laboratory manual . Cold Spring Harbor Laboratory Press , Cold Spring Harbor, N . Y . , 2001 ) and are well known to the skilled person in this field .
  • hybridisation temperature may be controlled, higher temperatures providing more stringent conditions .
  • Hybridisations performed at 42°C or higher may be considered to be under high stringency conditions .
  • both the temperature and washing conditions may be varied to provide hybridisation conditions of desired stringency .
  • probe hybridisation may be used to detect a BORIS nucleic acid in a sample, e . g . by northern blot (hybridising to mRNA) or southern blot (hybridising to DNA) .
  • Quantitative blotting techniques may be used to provide a relative assessment of the amount of BORIS nucleic acid detected .
  • the quantitative assessment may be used to assess the change in BORIS presence or expression and/or to indicate a change in the state of the cancerous condition, e . g . an increase or decrease in the malignancy of the cancer .
  • nucleic acid amplification based methods such as the Polymerase Chain Reaction (PCR)
  • PCR Polymerase Chain Reaction
  • One, two or more probes of predetermined length and having a sequence complementary or substantially complementary to a BORIS nucleic acid or fragment thereof along the entire length of the probe may be provided for use in nucleic acid amplification techniques to detect the presence and/or expression of BORIS nucleic acid in a patient sample .
  • the probes may be of any selected length, e . g . any of those indicated above in respect of fragments of nucleotide sequences .
  • kits provided for the diagnosis or prognosis of a cancerous condition .
  • kits may contain one or more anti-BORIS antibodies , optionally together with a secondary antibody capable of binding to the anti-BORIS antibody.
  • the secondary antibody may be linked to a visualisation agent .
  • Suitable visualisation agents are known to the person skilled in the art . For example, they may comprise fluorescent- or radio- labels .
  • a kit may contain one or more nucleic acid probes as described above for use in the detection of BORIS nucleic acid, e . g. BORIS mRNA.
  • the probes may be selected and/or designed to hybridise to a selected BORIS nucleic acid .
  • the hybridised probes may form primers for chain extension as part of a nucleic acid amplification technique such as PCR .
  • the probes may be linked to a visualisation agent . Suitable visualisation agents are known to the person skilled in the art . For example, they may comprise fluorescent- or radio- labels .
  • kits described may further comprise instructions for the use of the kit in the detection of BORIS nucleic acid or polypeptide as part of a method of diagnosis or prognosis of a cancerous condition .
  • the instructions may describe the use of the kit in the in vitro detection of BORIS nucleic acid or polypeptide from a blood sample or blood derived sample and may describe detection from a selected population of blood cells , such as PBCs .
  • a sample may be taken from any tissue or bodily fluid .
  • the sample is taken from a bodily fluid, more preferably one that circulates through the body .
  • the sample may be a blood sample or lymph sample .
  • the sample is a blood sample or blood-derived sample .
  • the blood derived sample may be a selected fraction of a patient' s blood, e . g . a selected cell-containing fraction or a plasma or serum fraction .
  • a selected cell-containing fraction may contain cell types of interest which may include white blood cells (WBC) , particularly peripheral blood mononuclear cells (PBC) and/or granulocytes , and/or red blood cells (RBC) . Accordingly, methods according to the present invention may involve detection of a BORIS polypeptide or nucleic acid in the blood, in white blood cells , peripheral blood mononuclear cells , granulocytes and/or red blood cells .
  • a selected serum fraction may comprise the fluid portion of the blood obtained after removal of the fibrin clot and blood cells .
  • the sample may be taken from blood precursor tissues , such as blood cell progenitor cells , circulating hemopoietic progenitors in blood such as granulocyte-macrophage precursor cells (CFU-GM) and erythroid precursor cells (BFU-E) , bone marrow or spleen .
  • blood precursor tissues such as blood cell progenitor cells , circulating hemopoietic progenitors in blood such as granulocyte-macrophage precursor cells (CFU-GM) and erythroid precursor cells (BFU-E) , bone marrow or spleen .
  • CFU-GM granulocyte-macrophage precursor cells
  • BFU-E erythroid precursor cells
  • the sample may comprise or may be derived from a tissue sample, biopsy or isolated cells from said individual .
  • the quantity of material contained in the sample should be sufficient to permit analysis of the sample according to the methods of the present invention .
  • a cancerous condition may be any unwanted cell proliferation, neoplasm or tumour or increased risk of or predisposition to an unwanted cell proliferation, neoplasm or tumour .
  • the cancerous condition may be any cancer and may be a benign or malignant cancer .
  • a neoplasm or tumour may be any abnormal growth or proliferation of cells and may be located in any tissue .
  • tissues where neoplasms or tumours may occur include the pancreas, lung, breast, ovaries , uterus , stomach, kidney, testis , central nervous system (including the brain) , peripheral nervous system, skin, blood or lymph .
  • the cancerous condition may be a cancer of any one of these tissues .
  • the cancerous condition is breast cancer .
  • the patient may be any animal or human .
  • the patient may be a non-human mammal , but is more preferably a human patient .
  • the patient may be male or female .
  • methods according to the present invention may be directed to diagnosis of cancerous conditions or assessment of the effectiveness of treatment of a cancerous condition in female patients .
  • Detection in a sample of BORIS polypeptides or nucleic acids in accordance with the methods of the present invention may be used for the purpose of diagnosis of a cancerous condition in the patient, diagnosis of a predisposition to a cancerous condition or for providing a prognosis (prognosticating) of a cancerous condition .
  • the diagnosis or prognosis may relate to an existing (previously diagnosed) cancerous condition, which may be benign or malignant, may relate to a suspected cancerous condition or may relate to the screening for cancerous conditions in the patient (which may be previously undiagnosed) .
  • diagnostic tests may be used in conjunction with those described here to enhance the accuracy of the diagnosis or prognosis or to confirm a result obtained by using the tests described here .
  • nucleic acid sequences may be identified by using hybridization and washing conditions of appropriate stringency.
  • Complementary nucleic acid sequences will hybridise to one another through Watson-Crick binding interactions . Sequences which are not 100% complementary may also hybridise but the strength of the hybridisation usually decreases with the decrease in complementarity. The strength of hybridisation can therefore be used to distinguish the -degree of complementarity of sequences capable of binding to each other .
  • the "stringency" of a hybridization reaction can be readily determined by a person skilled in the art .
  • the stringency of a given reaction may depend upon factors such as probe length, washing temperature, and salt concentration . Higher temperatures are generally required for proper annealing of long probes , while shorter probes may be annealed at lower temperatures . The higher the degree of desired complementarity between the probe and hybridisable sequence, the higher the relative temperature which can be used. As a result, it follows that higher relative temperatures would tend to make the reaction conditions more stringent, while lower temperatures less so .
  • hybridizations may be performed, according to the method of Sambrook et al . , ("Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory Press , 1989 ) using a hybridization solution comprising : 5X SSC, 5X Denhardt ' s reagent, 0.5-1.0% SDS, 100 ⁇ g/ml denatured, fragmented salmon sperm DNA, 0.05% sodium pyrophosphate and up to 50% formamide . Hybridization is carried out at 37-42 °C for at least six hours .
  • filters are washed as follows : (1 ) 5 minutes at room temperature in 2X SSC and 1% SDS; (2 ) 15 minutes at room temperature in 2X SSC and 0.1% SDS; (3 ) 30 minutes-1 hour at 37 °C in IX SSC and 1% SDS; ( 4 ) 2 hours at 42-65°C in IX SSC and 1% SDS, changing the solution every 30 minutes .
  • T m 81.5 °C + 16.6Log [Na+] + 0.41 (% G+C) - 0.63 (% formamide) - 600/n where n is the number of bases in the oligonucleotide .
  • the T n is 57 0 C .
  • the T m of a DNA duplex decreases by 1 - 1.5 °C with every 1% decrease in sequence complementarity .
  • nucleotide sequences can be categorised by an ability to hybridise to a target sequence under different hybridisation and washing stringency conditions which can be selected by using the above equation .
  • the T m may be used to provide an indicator of the strength of the hybridisation .
  • Sequences exhibiting 95-100% sequence complementarity are considered to hybridise under very high stringency conditions , sequences exhibiting 85-95% complementarity are considered to hybridise under high stringency conditions, sequences exhibiting 70-85% complementarity are considered to hybridise under intermediate stringency conditions, sequences exhibiting 60-70% complementarity are considered to hybridise under low stringency conditions and sequences exhibiting 50-60% complementarity are considered to hybridise under very low stringency conditions .
  • percentage (%) sequence identity of peptide or polypeptide sequences is defined as the percentage of amino acid residues in a candidate sequence that are identical with residues in the given sequence listed in this specification (which may be referred to by the SEQ ID No . ) after aligning the sequences and introducing gaps if necessary, to achieve the maximum sequence identity, and not considering any conservative substitutions as part of the sequence identity. Sequence identity is preferably calculated over the entire length of the respective sequences .
  • sequence identity of the shorter sequence is determined over the entire length of the longer sequence .
  • the candidate sequence can only have a maximum identity of 10% to the entire length of the given sequence . This is further illustrated in the following examples :
  • Alignment for purposes of determining percent amino acid sequence identity can be achieved in various ways known to a person of skill in the art, for instance, using publicly available computer software such as BLAST, BLAST-2 , ALIGN or Megalign (DNASTAR) software .
  • Identity of nucleic acid sequences may be determined in a similar manner involving aligning the sequences and introducing gaps if necessary, to achieve the maximum sequence identity, and calculating sequence identity over the entire length of the respective sequences . Where the aligned sequences are of different length, sequence identity of the shorter sequence is determined over the entire length of the longer sequence .
  • BORIS polypeptide and nucleic acid sequences Whilst detection of full length BORIS polypeptide and nucleic acid sequences according to any one of Figures 11 to 13 may be the subj ect of the methods of the present invention, this is not always necessary . As an alternative, detection of BORIS homologues, mutants, derivatives or fragments of these sequences may be used.
  • Derivatives include variants of a given full length amino acid or nucleic acid sequence and include naturally occurring allelic variants having substantial sequence identity to a sequence as set out in any one of Figures 11 to 13.
  • Mutants may comprise at least one modification (e . g . addition, substitution, inversion and/or deletion) compared to the corresponding wild-type . Mutations may occur in any of the sequences set out in Figures 11 to 13.
  • Derivatives may also comprise natural variations or polymorphisms which may exist between individuals or between members of a family. All such derivatives are included within the scope of the invention . Purely as examples , conservative replacements which may be found in such polymorphisms may be between amino acids within the following groups : (i ) alanine, serine, threonine; (ii) glutamic acid and aspartic acid; (iii ) arginine and leucine;
  • BORIS is a candidate blood borne tumour marker for cancer, e . g . breast cancer.
  • a circulating tumour marker of this kind nay be extremely useful clinically in a number of situations , such as :
  • mammography is presently the main tool used for breast cancer screening .
  • Screening mammography results in early detection and treatment, and a 30-33% reduction in mortality in women over the age of 50.
  • no clinically useful circulating tumour marker for this purpose has been identified.
  • Diagnosis of cancer For example, as a tumour marker that distinguishes benign from malignant processes . 4 . Prognosis .
  • Detection of BORIS in components of the blood provides the basis for an easily performed blood test that reliably predicts prognosis, independent of the stage of cancer .
  • prognostic markers for breast cancer are important because a significant number of patients with early stage breast cancer harbour microscopic metastasis at the time of diagnosis .
  • adjuvant systemic therapy improves survival in patients with early-stage breast cancer .
  • systemic therapies such as chemotherapy and hormone therapy, are potentially toxic and, unfortunately, histologic information is not sufficient to accurately assess individual risk and to possibly avoid adjuvant systemic therapy .
  • Tamoxifen reduces the risk of breast cancer in women at high risk for the disease but increases the risk for endometrial tumors and venous thromboembolisms . Therefore, identifying individual patients that are at high risk of developing a cancer and are likely to benefit from continued prognostic assessment provides a significant advantage in monitoring, detecting and applying appropriate and proportionate treatment for cancer at an early stage .
  • Prediction of response to therapy provides an indication of whether a specific therapy will benefit a patient .
  • the only current predictive factor is the presence of estrogen receptors in breast cancer tissues to predict a high probability of response to hormone therapy .
  • Providing a predictive factor is especially useful in breast cancer, since so many therapies are available with widely differing toxicities .
  • Aptamers are nucleic acid molecules characterised by the ability to bind to a target molecule with high specificity and high affinity .
  • Aptamers to a given target may be identified by the method of Systematic Evolution of Ligands by Exponential enrichment ( SELEXTM) . Aptamers and SELEX are described in WO91/19813.
  • Aptamers may be DNA or RNA molecules and may be single stranded or double stranded.
  • the aptamer may comprise chemically modified nucleic acids, for example in which the sugar and/or phosphate and/or base is chemically modified.
  • Such modifications may improve the stability of the aptamer or make the aptamer more resistant to degradation and may include modification at the 2' position of ribose .
  • Aptamers can be thought of as the nucleic acid equivalent of monoclonal antibodies and often have K d ' s in the nM or pM range . As with monoclonal antibodies , they may be useful in virtually any situation in which target binding is required, including use in therapeutic and diagnostic applications, in vitro or in vivo . In vitro diagnostic applications may include use in detecting the presence or absence of a target molecule .
  • an aptamers may be used in place of an antibody in any of the methods described here .
  • the invention includes the combination of the aspects and preferred features described except where such a combination is clearly impermissible or expressly avoided.
  • FIG. 1 Western blot showing frequency of abnormal BORIS activation in primary breast tumours .
  • IDC Invasive Ductal Carcinoma
  • DCIS Ductal Carcinoma in-situ
  • ILC Infiltrating Lobular Carcinoma
  • LCIS Lobular Carcinoma in-situ
  • LHP Lobular Hyperplasia
  • AD Adenocarcinoma
  • MD Medullary Carcinoma .
  • FIG. 1 Micrographs of immununohistochemical (IHC) staining of normal breast tissue (left hand panel) and breast tumour tissue (right hand panel ) samples with haematoxylin and anti- BORIS antibodies . Arrows indicate BORIS staining.
  • IHC immununohistochemical
  • Figure 3 Table showing breast tumour tissues tested, associated staining intensity and percentage (% ) of total cells tested expressing BORIS . Abbreviations follow those in Figure 1.
  • FIG. 4 Micrographs showing immununohistochemical (IHC) staining of the blood mononuclear layer of a normal , non- cancer, patient (left hand panel ) and the blood mononuclear layer of a patient with breast cancer (right hand panel) . Samples were stained with haematoxylin and anti-BORIS antibodies .
  • Figure 5. Micrographs showing iramununohistochemical ( IHC) staining of the blood mononuclear layer of a normal, non- cancer, patient (upper panels) and the blood mononuclear layer of a patient with breast cancer (lower panels ) . Samples were either stained with haematoxylin and anti-BORIS antibodies (left hand panels ) or with haematoxylin and a secondary antibody as a control (right hand panels ) .
  • Figure 6 Result of western blot analysis of white blood cell populations ( IxIO 5 cell/lane) from cancer cell patients and a normal , non-cancer patient (N) . Samples were probed with anti-BORIS antibody and show the presence of BORIS polypeptide in samples from cancer patients , but not in the normal (N) sample .
  • peripheral blo ⁇ d mononuclear cells PBC
  • IRS immunoreactivity scoring system.
  • FIG. 8 Table showing results of immunostaining of peripheral blood mononuclear cells (PBC) fr.om breast cancer patients with anti-BORIS antibody and correlation with patient tumour phenotype .
  • IRS immunoreactivity scoring system
  • IDC Invasive ductal carcinoma
  • ILC invasive lobular carcinoma
  • IADC invasive adenocarcinoma
  • ITC invasive tubular carcinoma
  • IDAC invasive ductal adenocarcinoma
  • DC ductal carcinoma
  • DCIS ductal carcinoma in situ
  • LCIS lobular carcinoma in situ
  • IP intraductal papilloma
  • ASC Angiosarcoma
  • DE ductal ectasia and N . D. , no details .
  • FIG. 9 Graph showing IRS value of BORIS in blood versus tumour size . IRS, immunoreactivity scoring system.
  • Figure 10. Graph showing IRS value of BORIS in blood versus tumour grade . IRS, iitimunoreactivity scoring system.
  • SEQ ID No . l represents the nucleotide sequence corresponding to human BORIS .
  • SEQ ID No .3 represents the nucleotide sequence corresponding to murine BORIS .
  • SEQ ID No .2 represents the amino acid sequence corresponding to human BORIS polypeptide .
  • SEQ ID No .4 represents the amino acid sequence corresponding to murine BORIS polypeptide .
  • Figure 13 Extract from NCBI database showing the entry for accession number NM_080618 (GI : 29570784 ) illustrating amino acid and nucleic acid sequence information for BORIS (SEQ ID No . s 5 and 6 respectively) .
  • FIG. 14 Examination of BORIS expression in myeloid leukemia and multiple myeloma cells by Western assay and immunofluorescent staining .
  • A Western blot analysis ;
  • B Preparations of KMMl and K562 cells were antibody-specific for BORIS and CTCF and reacted with DAPI .
  • BORIS expression is aberrantly activated in cancer cell lines .
  • BORIS as a potential cancer biomarker Validation studies Materials and Methods
  • PBC peripheral blood cells
  • the first step of preparation of PBC involved separation of PBC from serum.
  • 3 ml of histopaque was added with pastette to a 15 ml falcon tube .
  • the whole blood was pipetted and 7 ml carefully layered onto the gradient on side of the tube, in order not to mix the blood with the histopaque, centrifuged at 2500 rpm for 20 itiin .
  • Fluid serum
  • the cells in the 1.5 ml interface (PBC) were collected and transferred into a new tube .
  • the PBC were then washed with 1 X PBS ( 0.01M phosphate buffer, 0.0027M KCl and 0.137 NaCl, pH 7.4) and centrifuged for 5 min at 1500 rpm. Washes were repeated 3 times , the pellet was then re- suspended in 400 ⁇ l of 1 X PBS, transferred in 1.5 ml eppendorf tubes and cells counted and plated into 8-well chamber slide at ⁇ 2.5 10 4 cells/well . Cells were then fixed with 4% formaldehyde for 30 min, washed twice with 1 X PBS and stored in the fridge in 1 X PBS supplemented with 0.01% sodium azide . The remaining cells were centrifuged at 2 , 000 rpm for 5 min, the supernatant removed and the pellet dissolved in the loading buffer for Western (7M
  • the slides with PBC were placed in a stainless steel slide rack and fixed in acetone at room temperature (RT) for 15 minutes in a destaining j ar .
  • the slides were washed three times in 1 x PBS for 10 minutes on a shaking platform at RT (washing procedure) .
  • the excess PBS was drained off and the slides were quenched in a Coplin jar containing RT methanol and 1.6% H 2 O 2 ( 30% w/w (Sigma) ) for 20 minutes at RT on a shaking platform.
  • the slides were washed in 1 x PBS as per the washing procedure described above . After removal of the excess PBS, the slides were added to a Coplin jar containing 1 x PBS and 0.2% Triton X 100 (Sigma) and incubated at RT for 20 minutes on a shaking platform. Again, the slides were washed in PBS, as before . The sections were then blocked using 10% heat inactivated Normal
  • the sections were counterstained using VECTOR ® Haematoxylin QS (Vector Laboratories; H-3404 ) . This was dripped onto each section for 6 seconds and then indirectly washed with running tap water . The slides were then dehydrated twice in 95% and 100% ethanol for 2 minutes and followed by xylene . The slides were slightly dried and then mounted in VECTASHEILD ® Mounting Medium (Vector Laboratories; H-1000) .
  • Immunological staining was evaluated by using the immunoreactivity score (IRS) as previously described 4 .
  • the percentage of CTCF-positive cells was divided into four categories ( ⁇ 10% , 11-50% , 51-80% and >80% ) , whereas the staining intensity was given a scale from 0 (no detectable immunostaining) to 3 (strong immunostaining) .
  • the IRS ( 0-12) was then calculated by multiplying the score values .
  • Cells were plated into 8-well chamber slide at ⁇ 2.5 10 4 cells/ well , washed for a few seconds in 1 X PBS and fixed in 4% formaldehyde for 30 min at RT . Fixed cells were then washed twice with 1 X PBS and microwaved, at full cook mode, for 5 min in 10 itiM Citrate Buffer pH 6.0. Cells were then washed for 5 min in 1 X PBS , permeabilized with 1 X PBS / 0.2% Triton X-100 for 20 min, washed for 10 min with 1 X PBS and blocked in 1 X PBS - 2% HI-FBS (Heat Inactivated ( at 56°C for 30 min) Fetal Bovine Serum) for 2-3 hours .
  • 1 X PBS permeabilized with 1 X PBS / 0.2% Triton X-100
  • Samples from the frozen tissue sections were homogenized in the lysis buffer (2OmM Tris/Hepes pH 8.0, 2itiM EDTA, 0.5M NaCl , 0.5% Na deoxycholate, 0.5% Triton X-100, 0.25M Sucrose, 5OmM ⁇ - mercaptoethanol , and protease inhibitor kit (Roche) ) at approximate ratio of 5mg of tissue/ml buffer, the extract incubated on ice for 20 min, filtered, centrifuged at 14 , 000 rpm at +4 0 C and the supernatant collected.
  • the lysis buffer 2OmM Tris/Hepes pH 8.0, 2itiM EDTA, 0.5M NaCl , 0.5% Na deoxycholate, 0.5% Triton X-100, 0.25M Sucrose, 5OmM ⁇ - mercaptoethanol , and protease inhibitor kit (Roche)
  • Total cell lysates prepared for loading in sodium dodecyl sulfate (SDS ) -containing buffer with brief treatment with DNAse I to reduce viscosity (optional ) , were run through SDS-10% polyacrylamide gel electrophoresis (PAGE) gels and transferred onto ImmobilonTM P membranes (MilliporeTM, Bedford, Mass . , USA) by semi-dry blotting . Membranes were then probed with the appropriate antibody .
  • the molecular weight markers incorporated in the electrophoresis experiment were the pre-stained protein marker, broad range (premixed format) , P7708S New England BioLabsTM (www . neb. com or www. neb . com/neb/msds/new/P7708.pdf) .
  • Anti BORIS antibodies used were affinity purified chicken IgY developed against N terminal sequence of BORIS as described previously 1 .
  • Antibody solution is buffered with 5OmM KH 2 PO 4 , 20% glycerol and 1% BSA.
  • the working titer for western was 1/1000 , and for in situ immunostaining was 1 /25.
  • Specificity of the anti-BORIS antibodies was demonstrated by examination of BORIS expression in myeloid leukemia and multiple myeloma cells by Western assay and immunofluorescent staining ( Figure 14 ) .
  • FIG. 14A For western analysis ( Figure 14A) , cellular extracts from a myeloid leukemia cell line K562 and three multiple myeloma cell lines KMMl , KMS-5 and H929 positive for BORIS by RT-PCR were prepared from 5xlO 6 cells, total protein concentration was determined for each sample and equal amount ( 40 ⁇ g) of total protein loaded onto SDS-PAGE . Samples were electrophoretically separated, blotted and subsequently probed with the antibodies specific for BORIS and CTCF . The same membrane was stripped and re-probed with the anti- ⁇ -tubulin antibody (loading control ) . Preparations of KMMl and K562 cells were antibody-specific for BORIS and CTCF and reacted with DAPI ( Figure 14B) .
  • Anti-BORIS antibody specificity was further demonstrated by western blot in cancer cell lines in which BORIS expression is aberrantly activated ( Figure 15) .
  • Western blot analysis was performed on 28 cell lines obtained from the National Cancer Institute (NCI ) http : //www . nci . nih . gov/ .
  • Cellular extracts were prepared from 5xlO 6 cells , total protein concentration was determined for each sample and equal amount ( 40 ⁇ g) of total protein loaded onto SDS-PAGE . Samples were electrophoretically separated, blotted and probed with the antibodies specific for BORIS . The same membrane was stripped and re-probed with the anti- ⁇ -tubulin antibody (loading control ) .
  • the frequency of abnormal BORIS activation in primary breast tumours was investigated in 54 invasive ductal carcinomas , 5 lobular carcinomas , 8 ductal carcinomas in situ and paired adj acent normal breast tissue samples collected after surgery . Detection of the presence of BORIS in samples was by Western blot using anti-BORIS antibodies ( Figure 1 ) . BORIS was found to be expressed in 90% of these tumours .
  • the frequency of abnormal BORIS activation was further assessed by immunohistochemical (IHC) staining of samples of normal breast tissue and of breast tissue from patients with breast tumour . Staining was with haematoxylin and anti-BORIS antibodies ( Figure 2 ) . The presence of BORIS was detected in breast tissue from patients with breast tumour .
  • IHC immunohistochemical
  • IDC invasive ductal carcinoma
  • DCIS ductal carcinoma in situ
  • ILC invasive lobular carcinoma
  • ILCIS invasive lobular carcinoma in situ
  • AC adenocarcinoma
  • LLBH lobular hyperplasia benign
  • MD medullary carcinoma
  • BORIS polypeptide was detected in all samples from cancer patients tested but not in control samples from normal (N) , non-cancer, patients ( Figure 6) .
  • Figure 8 shows the correlation of IRS score with patient tumour phenotype .
  • the mean IRS value was 0.930. It is clear from Figure 8 that a majority of the tumour samples tested showed an increased IRS score over the normal mean value .
  • tumour grade is an indicator of the tumour' s malignancy based on the appearance of the tumour under the microscope and is an indication of how aggressive the cancer is .
  • Pathologists grade tumours by making an approximate assessment of the degree of structural de-differentiation by microscopic examination of tumours . The most differentiated tumours are Grade I and the most anaplastic are Grade IV, with Grades II and III lying in between .
  • a prognostic formula called The Nottingham Prognostic Index was used as a grading system in this study .
  • the inventors used anti-BORIS antibodies to investigate the presence of BORIS in peripheral blood cells (PBC) from breast cancer patients and normal individuals .
  • BORIS has been found to be aberrantly expressed in the granulocyte population of white blood cells from breast cancer patients .
  • Correlation of the BORIS IRS scores with important clinicopathological factors in primary breast cancer may also be of importance in helping reach an accurate diagnosis .
  • Patient information such as diagnosis (histopathology) , tumour stage, tumour grade, tumour size, steroid receptor (estrogen and progesterone ) status, lymph node metastasis, type of pre-operative and post-operative chemotherapy, menopausal state and age may be used in combination with the detection of the presence or absence of BORIS to provide a diagnosis .
  • diagnosis histopathology
  • tumour stage tumour grade
  • tumour size steroid receptor (estrogen and progesterone ) status
  • lymph node metastasis type of pre-operative and post-operative chemotherapy
  • menopausal state and age may be used in combination with the detection of the presence or absence of BORIS to provide a diagnosis .
  • breast tumour size and tumour grade emerged as very significant showing direct correlation with IRS in PBC .
  • BORIS as a cancer biomarker
  • the value of BORIS as a cancer biomarker is particularly important due to its presence in the circulating blood where it can be measured without using painful invasive procedures .

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Abstract

L'invention concerne des procédés de détection d'états cancéreux ou de prédisposition à de tels états, consistant à détecter un polypeptide ou un acide nucléique BORIS ou un fragment de ceux-ci. L'invention concerne également des procédés de diagnostic et de pronostic et/ou de pronostication d'un état cancéreux ou d'une prédisposition à un tel état.
PCT/GB2006/000364 2005-02-03 2006-02-03 Detection et diagnostic du cancer WO2006082414A1 (fr)

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CN101603068B (zh) * 2009-06-23 2012-04-18 山东省医学科学院基础医学研究所 苏木素在测定细胞增殖活性和药物对细胞毒效应中的应用
CN101650364B (zh) * 2009-07-31 2012-09-05 山东省医学科学院基础医学研究所 苏木素在检测免疫效应细胞介导的细胞毒效应中的应用
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LOUKINOV D I ET AL: "BORIS (BROTHER OF REGULATOR OF IMPRINTED SITES): A NOVEL TESTIS-SPECIFIC PARALOGUE OF CTCF AT 16Q22- MAPS TO A CANCER ASSOCIATED CHROMOSOME 20Q13 GAIN-AMPLIFICATION REGION, SHOWS ABERRANTLY ACTIVATED EXPRESSION IN MANY TYPES OF MALIGNANCIES, AND TRANSFORMS CELLS IN CULTURE", PROCEEDINGS OF THE ANNUAL MEETING OF THE AMERICAN ASSOCIATION FOR CANCER RESEARCH, NEW YORK, NY, US, vol. 44, July 2003 (2003-07-01), pages 1317, XP001204631, ISSN: 0197-016X *

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101603068B (zh) * 2009-06-23 2012-04-18 山东省医学科学院基础医学研究所 苏木素在测定细胞增殖活性和药物对细胞毒效应中的应用
CN101650364B (zh) * 2009-07-31 2012-09-05 山东省医学科学院基础医学研究所 苏木素在检测免疫效应细胞介导的细胞毒效应中的应用
US10415094B2 (en) 2016-10-06 2019-09-17 HelicalHelp LLC Risk stratification method for a patient having a polymorphism

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