WO2013040071A2 - Sperm protein as a detection biomarker of early stage ovarian cancer - Google Patents

Sperm protein as a detection biomarker of early stage ovarian cancer Download PDF

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WO2013040071A2
WO2013040071A2 PCT/US2012/054914 US2012054914W WO2013040071A2 WO 2013040071 A2 WO2013040071 A2 WO 2013040071A2 US 2012054914 W US2012054914 W US 2012054914W WO 2013040071 A2 WO2013040071 A2 WO 2013040071A2
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patient
gene
ovarian cancer
level
expression level
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PCT/US2012/054914
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English (en)
French (fr)
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WO2013040071A3 (en
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Wijbe Martin Kast
Maurizio Chiriva-Internati
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University Of Southern California
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Priority to JP2014530760A priority Critical patent/JP2014527816A/ja
Priority to US14/345,208 priority patent/US20150110897A1/en
Priority to CN201280054743.6A priority patent/CN104039980A/zh
Priority to IN2496CHN2014 priority patent/IN2014CN02496A/en
Priority to EP12831144.6A priority patent/EP2756100A4/en
Priority to CA 2848578 priority patent/CA2848578A1/en
Publication of WO2013040071A2 publication Critical patent/WO2013040071A2/en
Publication of WO2013040071A3 publication Critical patent/WO2013040071A3/en

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    • 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
    • G01N33/57407—Specifically defined cancers
    • G01N33/57449—Specifically defined cancers of ovaries
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P15/00—Drugs for genital or sexual disorders; Contraceptives
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • 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
    • G01N33/57473—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving carcinoembryonic antigen, i.e. CEA
    • 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
    • C12Q2600/118—Prognosis of disease development
    • 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
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/46—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans from vertebrates
    • G01N2333/47—Assays involving proteins of known structure or function as defined in the subgroups
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/705—Assays involving receptors, cell surface antigens or cell surface determinants

Definitions

  • Applicants analyzed SP17 expression in primary ovarian tissues from ovarian cancer patients, benign ovarian lesions and healthy subjects through RT-PCR and immunohistochemistry. SP17 and CA125 levels were measured in the sera of the same subjects by ELISA assays. From this analysis, this disclosure provides a method for determining if a patient is likely to, or not likely to, experience ovarian cancer, (i.e.
  • the method further comprises, or alternatively consists essentially of, or yet further consists of, determining the expression level of the CA125 gene in a sample isolated from the patient, wherein the presence of a SP17 level and a CA125 level or SP17 and CA125 levels higher than a predetermined first value identifies the patient as more likely to experience ovarian cancer; and the presence of a CA125 level or a CA125 levels higher than a predetermined first value and the absence of an SP17 level or a SP17 level lower than the predetermined first value identifies the patient as not likely to experience ovarian cancer.
  • Also provided is a method for determining if an ovarian cancer patient is likely to, or not likely to, experience longer or shorter overall survival comprising, or alternatively consisting essentially of, or yet further consisting of, determining the expression level of the SP17 gene in a sample isolated from the patient, wherein the presence of a SP17 gene expression level or a lever higher than a predetermined first value identifies the patient as more likely to experience shorter overall survival and the absence of SP17 gene expression or SP17 gene expression lower than the predetermined first value identifies the patient as likely to experience longer overall survival.
  • Figures 1A and IB show SP17 expression in primary ovarian cancer cells.
  • Figure 1 A PCR was performed in different samples to analyze SP17 presence in normal ovaries, benign ovarian tumor conditions, ovarian cancer patients and in a Skov-3 ovarian cancer cell line.
  • compositions and methods include the recited elements, but not excluding others.
  • Consisting essentially of when used to define compositions and methods shall mean excluding other elements of any essential significance to the composition or method.
  • Consisting of shall mean excluding more than trace elements of other ingredients for claimed compositions and substantial method steps. Embodiments defined by each of these transition terms are within the scope of this invention. Accordingly, it is intended that the methods and compositions can include additional steps and components (comprising) or alternatively including steps and compositions of no significance (consisting essentially of) or alternatively, intending only the stated method steps or
  • adjuvant cancer patient refers to a patient to which administration of a therapy or chemotherapeutic regimen has been given after removal of a tumor by surgery, usually termed adjuvant chemotherapy.
  • adjuvant therapy is typically given to minimize or prevent a possible cancer reoccurrence.
  • nonadjuvant therapy refers to administration of therapy or chemotherapeutic regimen before surgery, typically in an attempt to shrink the tumor prior to a surgical procedure to minimize the extent of tissue removed during the procedure.
  • S17 is a highly conserved mammalian protein in the testis and spermatozoa and has been characterized as a tumor-associated antigen in a variety of human malignancies.
  • wild-type allele refers to an allele of a gene which, when present in two copies in a subject results in a wild-type phenotype. There can be several different wild-type alleles of a specific gene, since certain nucleotide changes in a gene may not affect the phenotype of a subject having two copies of the gene with the nucleotide changes.
  • genotype refers to the specific allelic composition of an entire cell or a certain gene and in some aspects a specific polymorphism associated with that gene, whereas the term “phenotype” refers to the detectable outward manifestations of a specific genotype.
  • a "predetermined value" for a gene as used herein is so chosen that a patient with an expression level of that gene higher than the predetermined value is likely to experience a more or less desirable clinical outcome than patients with expression levels of the same gene lower than the predetermined value, or vice-versa.
  • Expression levels of genes are associated with clinical outcomes.
  • One of skill in the art can determine a predetermined value for a gene by comparing expression levels of a gene in patients with more desirable clinical outcomes to those with less desirable clinical outcomes.
  • a predetermined value is a gene expression value that best separates patients into a group with more desirable clinical outcomes and a group with less desirable clinical outcomes. Such a gene expression value can be mathematically or statistically determined with methods well known in the art.
  • Cells "host cells” or “recombinant host cells” are terms used interchangeably herein. It is understood that such terms refer not only to the particular subject cell but to the progeny or potential progeny of such a cell. Because certain modifications may occur in succeeding generations due to either mutation or environmental influences, such progeny may not, in fact, be identical to the parent cell, but are still included within the scope of the term as used herein.
  • the PCR procedure describes a method of gene amplification which is comprised of (i) sequence-specific hybridization of primers to specific genes within a DNA sample (or library), (ii) subsequent amplification involving multiple rounds of annealing, elongation, and denaturation using a DNA polymerase, and (iii) screening the PCR products for a band of the correct size.
  • the primers used are oligonucleotides of sufficient length and appropriate sequence to provide initiation of polymerization, i.e. each primer is specifically designed to be complementary to each strand of the genomic locus to be amplified.
  • a patient possessing a characteristic such as a genotype of a genetic polymorphism
  • the patients are considered suitable for the therapy.
  • a patient possessing a characteristic may exhibit one or more desirable clinical outcome but simultaneously exhibit one or more less desirable clinical outcome.
  • the clinical outcomes will then be considered collectively, and a decision as to whether the patient is suitable for the therapy will be made accordingly, taking into account the patient's specific situation and the relevance of the clinical outcomes.
  • progression free survival or overall survival is weighted more heavily than tumor response in a collective decision making.
  • a "complete response" (CR) to a therapy defines patients with evaluable but non- measurable disease, whose tumor and all evidence of disease had disappeared.
  • Tumor Recurrence as used herein and as defined by the National Cancer Institute is cancer that has recurred (come back), usually after a period of time during which the cancer could not be detected. The cancer may come back to the same place as the original (primary) tumor or to another place in the body. It is also called recurrent cancer.
  • Also provided is a method for determining if an ovarian cancer patient is likely to, or not likely to, experience longer or shorter overall survival comprising, or alternatively consisting essentially of, or yet further consisting of, determining the expression level of the SP17 gene in a sample isolated from the patient, wherein the presence of a SP17 gene expression level or a lever higher than a predetermined first value identifies the patient as more likely to experience shorter overall survival and the absence of SP17 gene expression or SP17 gene expression lower than the predetermined first value identifies the patient as likely to experience longer overall survival.
  • predetermined value is a gene expression value that best separates patients into a group with more desirable clinical parameter and a group with less desirable clinical parameter.
  • a gene expression value can be mathematically or statistically determined with methods well known in the art.
  • Methods to determine gene expression level are known in the art and briefly described herein. Non-limiting examples of these methods include a method that comprises, or alternatively consists essentially of, or yet further consists of, determining the amount of mRNA transcribed from the gene, mRNA in situ hybridization, PCR, real-time PCR, or microarray. The methods are useful in the assistance of a patient such as an animal, a mammal or yet further a human patient.
  • a mammal includes but is not limited to a simian, a murine, an ovine, an equine, a canine, a bovine, a porcine or a human patient.
  • Amplification can be performed, e.g., by PCR and/or LCR, according to methods known in the art.
  • PCR e.g., by PCR and/or LCR, according to methods known in the art.
  • Various non- limiting examples of PCR include the herein described methods.
  • PCR is carried out as usual, but with a great excess of the primers for the chosen strand. Due to the slow amplification later in the reaction after the limiting primer has been used up, extra cycles of PCR are required (See, Innis et al. (1988) Proc Natl Acad Sci U.S.A. 85(24):9436-9440 and U.S. Patent Nos.: 5,576,180; 6,106,777 or 7,179,600).
  • LATE-PCR Linear- After-The- Exponential-PCR
  • T m melting temperature
  • Colony PCR uses bacterial colonies, for example E. coli, which can be rapidly screened by PCR for correct DNA vector constructs. Selected bacterial colonies are picked with a sterile toothpick and dabbed into the PCR master mix or sterile water. The PCR is started with an extended time at 95 °C when standard polymerase is used or with a shortened denaturation step at 100°C and special chimeric DNA polymerase (Pavlov et al. (2006) "Thermostable DNA
  • MPA Multiplex Ligation-dependent Probe Amplification
  • Nested PCR increases the specificity of DNA amplification, by reducing background due to non-specific amplification of DNA.
  • Two sets of primers are being used in two successive PCRs. In the first reaction, one pair of primers is used to generate DNA products, which besides the intended target, may still consist of non-specifically amplified DNA fragments.
  • the product(s) are then used in a second PCR with a set of primers whose binding sites are completely or partially different from and located 3' of each of the primers used in the first reaction (See, U.S. Patent Nos.: 5,994,006; 7,262,030 or 7,329,493).
  • Nested PCR is often more successful in specifically amplifying long DNA fragments than conventional PCR, but it requires more detailed knowledge of the target sequences.
  • Overlap-extension PCR is a genetic engineering technique allowing the construction of a DNA sequence with an alteration inserted beyond the limit of the longest practical primer length.
  • RT-PCR refers to reverse transcription PCR (see below), which is often used in conjunction with Q-PCR.
  • QRT-PCR methods use fluorescent dyes, such as Sybr Green, or fluorophore-containing DNA probes, such as TaqMan, to measure the amount of amplified product in real time.
  • RT-PCR Reverse Transcription PCR
  • RACE-PCR Rapid Amplification of cDNA Ends
  • TAIL-PCR Thermal asymmetric interlaced PCR
  • Touchdown PCR a variant of PCR that aims to reduce nonspecific background by gradually lowering the annealing temperature as PCR cycling progresses.
  • the annealing temperature at the initial cycles is usually a few degrees (3-5 °C) above the T m of the primers used, while at the later cycles, it is a few degrees (3-5 °C) below the primer T m .
  • the higher temperatures give greater specificity for primer binding, and the lower temperatures permit more efficient amplification from the specific products formed during the initial cycles (Don et al. (1991) Nucl Acids Res 19:4008 and U.S. Patent No. 6,232,063).
  • probes are labeled with two fluorescent dye molecules to form so-called “molecular beacons” (Tyagi, S. and Kramer, F.R. (1996) Nat.
  • molecular beacons signal binding to a complementary nucleic acid sequence through relief of intramolecular fluorescence quenching between dyes bound to opposing ends on an oligonucleotide probe.
  • the use of molecular beacons for genotyping has been described (Kostrikis, L.G. (1998) Science 279: 1228-9) as has the use of multiple beacons simultaneously (Marras, S.A. (1999) Genet. Anal. 14: 151-6).
  • a quenching molecule is useful with a particular fluorophore if it has sufficient spectral overlap to substantially inhibit fluorescence of the fluorophore when the two are held proximal to one another, such as in a molecular beacon, or when attached to the ends of an oligonucleotide probe from about 1 to about 25 nucleotides.
  • This invention also provides for a prognostic panel of genetic markers selected from, but not limited to the genes of interest identified herein.
  • the prognostic panel comprises probes or primers that can be used to amplify and/or for determining the molecular structure of the SP17 alone or in combination with CA125.
  • the probes or primers can be attached or supported by a solid phase support such as, but not limited to a gene chip or microarray.
  • the probes or primers can be detectably labeled.
  • the panel contains the herein identified probes or primers as wells as other probes or primers.
  • the panel includes one or more of the above noted probes or primers and others.
  • the panel consist only of the above- noted probes or primers.
  • Primers or probes can be affixed to surfaces for use as "gene chips” or "microarray.” Such gene chips or microarrays can be used to detect genetic variations by a number of techniques known to one of skill in the art. In one technique, oligonucleotides are arrayed on a gene chip for determining the DNA sequence of a by the sequencing by hybridization approach, such as that outlined in U.S. Patent Nos. 6,025,136 and 6,018,041. The probes of the invention also can be used for fluorescent detection of a genetic sequence. Such techniques have been described, for example, in U.S. Patent Nos. 5,968,740 and 5,858,659.
  • a probe also can be affixed to an electrode surface for the electrochemical detection of nucleic acid sequences such as described by Kayem et al. U.S. Patent No. 5,952,172 and by Kelley et al. (1999) Nucleic Acids Res. 27:4830-4837.
  • BeadArray (Illumina, Inc.); GEM (Incyte Microarray Systems); a high-throughput
  • microarraying system that can dispense from 12 to 64 spots onto multiple glass slides
  • probes or primers for the gene of interest are provided alone or in combination with other probes and/or primers.
  • a suitable sample is obtained from the patient extraction of genomic DNA, RNA, or any combination thereof and amplified if necessary.
  • the DNA or RNA sample is contacted to the gene chip or microarray panel under conditions suitable for hybridization of the gene(s) of interest to the probe(s) or primer(s) contained on the gene chip or microarray.
  • the probes or primers may be detectably labeled thereby identifying the polymorphism in the gene(s) of interest.
  • a chemical or biological reaction may be used to identify the probes or primers which hybridized with the DNA or RNA of the gene(s) of interest.
  • the genetic profile of the patient is then determined with the aid of the aforementioned apparatus and methods.
  • Non- limiting examples of such include Adriamycin PFS; Adriamycin RDF; BEP and Carboplatin.
  • Therapeutic amounts can be empirically determined and will vary with the pathology being treated, the subject being treated and the efficacy and toxicity of the agent.
  • the invention provides diagnostic methods for determining the expression level of SP17 alone or in combination with CA125.
  • the methods use probes or primers comprising nucleotide sequences which are complementary to the gene of interest. Accordingly, the invention provides kits for performing these methods as well as instructions for carrying out the methods of this invention.
  • the components and instructions of the kit identifies a patient as more likely to experience tumor recurrence or shorter overall survival if SP17 expression level that is detected or is higher than the predetermined first value or alternatively, when CA125 and SP17 gene expression level that is present or higher than the predetermined values.
  • treatment protocols and/or therapies are further provided in the kit.
  • the components and instructions of the kit is used to diagnose ovarian cancer in a patient, comprising or alternatively consisting essentially of, or yet further consisting of, suitable reagents to perform the methods as described herein and instructions for use.
  • treatment protocols and/or therapies are further provided in the kit.
  • the components and instructions of the kit is used to determine if the patient as less likely to experience tumor recurrence or longer overall survival time when a SP17 gene expression is absent or lower than the predetermined first value, or if CA125 level is present or higher but SP17 level is absent or lower than the predetermined values.
  • a predetermined value is a gene expression value that best separates patients into a group with more desirable clinical parameters and a group with less desirable clinical parameters.
  • a gene expression value can be mathematically or statistically determined with methods well known in the art.
  • the kit can comprise at least one probe or primer which is capable of specifically hybridizing to the SP17 alone or in combination with probes and/or primer to determine the expression level of the CA125 gene and instructions for use.
  • the kits preferably comprise at least one of the above described nucleic acids.
  • Preferred kits for amplifying at least a portion of the gene of interest comprise two primers, at least one of which is capable of hybridizing to the allelic variant sequence.
  • Such kits are suitable for detection of genotype by, for example, fluorescence detection, by electrochemical detection, or by other detection.
  • Oligonucleotides whether used as probes or primers, contained in a kit can be detectably labeled. Labels can be detected either directly, for example for fluorescent labels, or indirectly. Indirect detection can include any detection method known to one of skill in the art, including biotin-avidin interactions, antibody binding and the like. Fluorescently labeled oligonucleotides also can contain a quenching molecule. Oligonucleotides can be bound to a surface. In one embodiment, the preferred surface is silica or glass. In another embodiment, the surface is a metal electrode.
  • kits of the invention comprise at least one reagent necessary to perform the assay.
  • the kit can comprise an enzyme.
  • the kit can comprise a buffer or any other necessary reagent.
  • Conditions for incubating a nucleic acid probe with a test sample depend on the format employed in the assay, the detection methods used, and the type and nature of the nucleic acid probe used in the assay.
  • One skilled in the art will recognize that any one of the commonly available hybridization, amplification or immunological assay formats can readily be adapted to employ the nucleic acid probes for use in the present invention. Examples of such assays can be found in Chard, T. (1986) AN INTRODUCTION TO RADIOIMMUNOASSAY AND
  • test samples used in the diagnostic kits include cells, protein or membrane extracts of cells, or biological fluids such as sputum, blood, serum, plasma, or urine.
  • the test sample used in the above-described method will vary based on the assay format, nature of the detection method and the tissues, cells or extracts used as the sample to be assayed. Methods for preparing protein extracts or membrane extracts of cells are known in the art and can be readily adapted in order to obtain a sample which is compatible with the system utilized.
  • kits can include all or some of the positive controls, negative controls, reagents, primers, sequencing markers, probes and antibodies described herein for determining the expression level of the gene of interest.
  • these suggested kit components may be packaged in a manner customary for use by those of skill in the art.
  • these suggested kit components may be provided in solution or as a liquid dispersion or the like.
  • Methods to determine gene expression level are known in the art and briefly described herein. Non-limiting examples of these methods include a method that comprises, or alternatively consists essentially of, or yet further consists of, determining the amount of mRNA transcribed from the gene, mRNA in situ hybridization, use of gene chips or microarray, PCR, real-time PCR, or microarray. The methods are useful in the assistance of a patient such as an animal, a mammal or yet further a human patient.
  • a mammal includes but is not limited to a simian, a murine, an ovine, an equine, a canine, a bovine, a porcine or a human patient.
  • the patients are stage 2 cancer patients and had not yet received any additional therapy after surgery or surgical resection.
  • the patients are stage 3 cancer patients and will receive or had received additional therapy after surgery or surgical resection.
  • BeadArray (Illumina, Inc.); GEM (Incyte Microarray Systems); a high-throughput
  • Reverse-transcription PCR was performed by standard techniques as previously described 19 ' 20 .
  • the primers for the SP17 transcript were 5'-GGA TCC ATG TCG ATT CCA TTC TC-3' (SEQ ID NO: 1) and 5' -CTC GAG TCA CTT GTT TTC CTC TTT TTC-3' (SEQ ID NO: 2).
  • Immunohistochemistrv 5'-GGA TCC ATG TCG ATT CCA TTC TC-3' (SEQ ID NO: 1) and 5' -CTC GAG TCA CTT GTT TTC CTC TTT TTC-3' (SEQ ID NO: 2).
  • tissue sections underwent antigen retrieval and staining as previously described 19 ' 21 .
  • Plates were washed thrice with PBS + 0.025% Tween-20 (PBS-t) and incubated with HRP-linked anti-mouse secondary antibody (Abeam, diluted 1 :4000 in PBS, 50 IJwell) for 2 hours. After 3 washing steps with PBS-t (300 ⁇ , ⁇ ), the chromogen substrate was added and allowed to incubate 5 minutes in the dark. Absorbance was then read at 405 nm. Serial dilutions of purified antigens were used to determine SP17 concentration in the serum.
  • HRP-linked anti-mouse secondary antibody Abeam, diluted 1 :4000 in PBS, 50 IJwell
  • CA125 was measured using the FDA-certified method ARCHITECT CA 125 IITM assay (Abbott Park, IL, USA), according to the manufacturer's directions.
  • Applicants studied a total of 323 subjects (136 ovarian cancer patients, 45 benign ovarian tumors and 142 age-matched healthy volunteers). Subjects' distribution was as follows: 13% stage I, 6% stage II, 20% stage III, 4% stage IV, 44% healthy, 13% benign ovarian tumors. 44% of ovarian cancer were grade I/II, and 55% were grade III. Median age of ovarian cancer patients, ovarian benign tumor patients and healthy controls was 59 years. SP17 is expressed at different levels in primary cells from healthy subjects, benign ovarian tumors, and ovarian cancer lesions.
  • SP 17 mR A was evaluated in normal ovaries, benign ovarian tumor samples, and ovarian cancer primary cells (Fig. 1 A). Positive bands were detectable in samples from ovarian cancer patients, but no transcript was present in the benign ovarian tumor samples and healthy controls. At the protein level, SP17 was detectable in all ovarian cancer samples ranging from stages I-IV, while no protein expression was shown in normal ovaries (Fig. IB), or in any of 45 analyzed benign ovarian lesions (Fig. IB).
  • Table I shows mean SP17 and CA125 serum concentration measured in healthy controls, benign ovarian tumor or ovarian cancer patients at diagnosis: mean SP17 and CA125 levels were significantly different between groups, as confirmed by one-way ANOVA. Dunn's Multiple Comparison Test revealed that SP17 and CA125 significantly lower in healthy and benign groups, as compared with ovarian cancer patients (Table I). No significant difference was detected between ovarian cancer stages, or between healthy and benign groups (p>0.05).
  • SP17 serum concentration is associated with overall survival.
  • SP17 and CA125 afforded similar AUC (p>0.05).
  • ovarian cancer screening is clinically validated only when the disease prevalence is reasonably high, that is for women carrying BRCAl/2 mutations or with a family history of breast or ovarian cancer .
  • (9) Since its discovery in 1981 as a potential biomarker to monitor the course of the disease, (22) CA125 measurement alone or together with transvaginal ultrasonography (TVU) has been widely explored as a diagnostic test for early detection of ovarian cancer, with limited success.
  • TVU transvaginal ultrasonography
  • Increasing efforts are being made for the identification of novel tumor biomarkers to improve CA125 test specificity and sensitivity.
  • SP17 will prove to be the best immunohisto logic tool to provide the highest prognostic index in ovarian tumors.
  • the finding that SP17 was selectively expressed only in ovarian cancer samples is consistent with previous studies, indicating that SP17 allows for tracking ovarian cancer in a murine model of the disease (14), and that it is expressed in 83% of primary ovarian tumors, but not by normal cells of the ovary.
  • Applicants investigated the presence of free circulating SP17 protein in the peripheral blood of ovarian cancer and benign ovarian tumor patients, or healthy controls. The data clearly indicated that serum SP17 levels were significantly higher in ovarian cancer than in healthy or benign groups.
  • a similar result was obtained for CA125, which was used as a reference thorough the present study, since it is a validated serologic prognostic indicator. (37, 38) This is the first time that SP17 protein was analyzed in the serum of healthy individuals and cancer patients.
  • Applicants show that the measurement of SP17 serum concentration by ELISA assay affords significant discrimination between subjects with different prognoses, specifically benign ovarian tumors and ovarian carcinomas, and Applicants provide evidence that SP17 serum level is a prognostic indicator of both progression-free and overall survival. Applicants show for the first time a serum biomarker suitable for diagnosis of early stage ovarian cancer that is potentially applicable in a large-scale screening.
  • Tumour-specific HMG-CoAR is an independent predictor of recurrence free survival in epithelial ovarian cancer.

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PCT/US2012/054914 2011-09-15 2012-09-12 Sperm protein as a detection biomarker of early stage ovarian cancer WO2013040071A2 (en)

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JP2014530760A JP2014527816A (ja) 2011-09-15 2012-09-12 初期卵巣がんの検出マーカーとしての精子タンパク質
US14/345,208 US20150110897A1 (en) 2011-09-15 2012-09-12 Sperm Protein as a Detection Biomarker of Early Stage Ovarian Cancer
CN201280054743.6A CN104039980A (zh) 2011-09-15 2012-09-12 精子蛋白作为早期卵巢癌的检测生物标记物
IN2496CHN2014 IN2014CN02496A (GUID-C5D7CC26-194C-43D0-91A1-9AE8C70A9BFF.html) 2011-09-15 2012-09-12
EP12831144.6A EP2756100A4 (en) 2011-09-15 2012-09-12 SPERM PROTEIN AS DETECTION BIOMARKER FOR THE EARLY DETECTION OF EGG CANCER
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CN104807997A (zh) * 2015-05-05 2015-07-29 南京格耀生物科技有限公司 一种基于化学发光法检测ca125和sp17含量的试剂盒及方法和应用

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US7405077B2 (en) * 2001-02-26 2008-07-29 Albany Medical College Sperm protein 17 for the diagnosis and treatment of cancer
US7189507B2 (en) * 2001-06-18 2007-03-13 Pdl Biopharma, Inc. Methods of diagnosis of ovarian cancer, compositions and methods of screening for modulators of ovarian cancer
AT501348B1 (de) * 2005-01-31 2008-10-15 Vitateq Biotechnology Gmbh Verfahren zur tumordiagnose
CN100334110C (zh) * 2005-09-01 2007-08-29 中国人民解放军南京军区南京总医院 一种精子蛋白单克隆抗体及其制备方法和用途
DE602006015508D1 (de) * 2005-11-16 2010-08-26 Pantarhei Bioscience Bv Pharmazeutische zusammensetzung zur behandlung oder prävention von ovarialkrebs

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* Cited by examiner, † Cited by third party
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CN104807997A (zh) * 2015-05-05 2015-07-29 南京格耀生物科技有限公司 一种基于化学发光法检测ca125和sp17含量的试剂盒及方法和应用

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CA2848578A1 (en) 2013-03-21
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