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WO2008061370A1 - Procédé de diagnostic permettant de détecter l'endométriose - Google Patents

Procédé de diagnostic permettant de détecter l'endométriose Download PDF

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WO2008061370A1
WO2008061370A1 PCT/CA2007/002114 CA2007002114W WO2008061370A1 WO 2008061370 A1 WO2008061370 A1 WO 2008061370A1 CA 2007002114 W CA2007002114 W CA 2007002114W WO 2008061370 A1 WO2008061370 A1 WO 2008061370A1
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trkb
endometriosis
sample
expression
endometrial
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Warren Foster
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McMaster University
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McMaster University
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    • 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
    • C12Q1/48—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving transferase
    • C12Q1/485—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving transferase involving kinase
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/095—Sulfur, selenium, or tellurium compounds, e.g. thiols
    • A61K31/105—Persulfides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • A61K31/713—Double-stranded nucleic acids or oligonucleotides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • 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
    • 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
    • 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
    • G01N2333/90—Enzymes; Proenzymes
    • G01N2333/91—Transferases (2.)
    • G01N2333/912—Transferases (2.) transferring phosphorus containing groups, e.g. kinases (2.7)
    • G01N2333/91205—Phosphotransferases in general
    • G01N2333/9121—Phosphotransferases in general with an alcohol group as acceptor (2.7.1), e.g. general tyrosine, serine or threonine kinases
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/36—Gynecology or obstetrics
    • G01N2800/364—Endometriosis, i.e. non-malignant disorder in which functioning endometrial tissue is present outside the uterine cavity

Definitions

  • the present invention relates to methods and compositions for detecting and monitoring endometrial disease, such as endometriosis and endometriosis associated ovarian cancer.
  • Endometriosis is an estrogen-dependent disease characterized by the growth of endometrial stromal cells and glands outside of the uterine cavity. Between 1 and 7% of women in the general population and up to 30% of women undergoing laparoscopy for chronic pelvic pain are diagnosed with endometriosis. Although the cause of endometriosis remains an enigma, retrograde menstruation of shed endometrial cells and tissue fragments is thought to play a role in the development of this disease. However, factors other than retrograde menstruation are thought to contribute to the pathogenesis of endometriosis.
  • Endometriosis is multifactorial in origin involving features of immune modulation, adhesion, angiogenesis, invasion, proliferation and decreased apoptosis.
  • the critical event(s) or biochemical change(s) that ultimately leads to establishment of endometriosis remains unknown.
  • endometrial cells destined to become endometriotic implants are biochemically and functionally distinct from eutopic endometrium of women without endometriosis. Endometrial cells from women with endometriosis survived transplantation in athymic nude mice for months whereas normal proliferative endometrial cells from women without disease did not implant and proliferate. The endometrium of women with endometriosis aberrantly express cell adhesion molecules such as integrins and cadherins. There is some evidence suggesting that an imbalance between proliferation and apoptosis signals in the endometrium may play a role in the development of endometriosis.
  • IL-6 interleukin-6
  • I L-6s R soluble receptor
  • EAOC endometriosis associated ovarian cancer
  • Endometriosis and malignancy was first reported to occur concurrently by Sampson in 1925. Since then, a number of studies have been conducted that have shed light on the characteristics of endometriosis associated ovarian cancer. A great proportion of EAOC cases present with either endometrioid carcinoma or clear cell carcinoma, although other types are possible. It has also been reported that women with EAOC are generally younger patients. According to epidemiological evidence, a noted risk factor for ovarian cancer includes endometriosis, further suggesting a relation between the two diseases.
  • Tropomyosin-related kinase receptors A, B and C are neutrophin receptors that regulate the proliferation, differentiation and programmed cell death of neuronal cells.
  • Trk activity in areas outside of the nervous system has also been put forth.
  • TrkA expression in pancreatic cancer has been shown to act as a marker of tumour aggressiveness and invasiveness.
  • TrkA plays a role in gynaecological pathologies.
  • Increased TrkA expression has been shown to be correlated with ovarian carcinoma tumour progression.
  • TrkA was found to be up regulated in advanced end stage ovarian carcinomas. Not only have the tyrosine kinase receptor A have been shown to be up regulated in various malignancies, evidence of over expression of their cognate ligand, nerve growth factor (NGF), have also been observed. TrkB is believed to be important in regulating malignant epithelial cancer cell resistance to anoikis; attachment free mediated programmed cell death. Although the underlying mechanism is not fully understood and remains unknown, it is believed that TrkB plays a role in tumourgenesis and metastasis of malignancies.
  • NGF nerve growth factor
  • Neurotrophic factors including brain derived neurotrophic factor (BDNF) and its cognate receptor tyrosine receptor kinase B (TrkB) have been localized in non-neural tissues and epithelial tumours.
  • BDNF brain derived neurotrophic factor
  • TrkB cognate receptor tyrosine receptor kinase B
  • TrkB cognate receptor tyrosine receptor kinase B
  • TrkB may consequently play an additional role in germ-cell survival and follicular maturation in mammalian ovaries. Therefore, neurotrophic factors signalling through TrkB could be important in regulation of reproductive tissue and tumour behaviour.
  • the present invention provides methods for the detection, prediction, and staging of endometrial diseases. Therapeutic compositions and methods are also provided.
  • endometrial disease refers to any disorder associated with aberrant proliferation or localization of endometrial cells. This includes endometriosis and endometriosis associated ovarian cancer (EAOC).
  • EAOC endometriosis and endometriosis associated ovarian cancer
  • TrkA, TrkB, and TrkC are the functional receptors for the neurotrophins nerve growth factor (NGF), brain derived neurotrophic factor (BDNF) neurotrophin-4/5 (NT-4/5), and neurotrophin-3 (NT-3), respectively.
  • Tyrosine kinase receptors are also aberrantly expressed in the endometrium of women with endometriosis and ovarian endometriosis.
  • TrkB is a neurotrophic receptor best known for its role in the nervous system where it regulates cellular proliferation, differentiation and survival.
  • TrkB expression in epithelial cancer cells was shown to be obligatory for matrix free escape from apoptosis. TrkB expression could be important in the survival of regurgitated endometrial cells and tissue fragments and thus its expression may be related to the development of endometriosis.
  • TrkB is expressed in many tissues throughout the human body including the ovary. Results of two gene array studies revealed the presence of the receptor in endometrial epithelial cells of women with endometriosis.
  • the present invention demonstrates a correlation between TrkB expression and endometriosis.
  • the expression of TrkB in archived paraffin embedded endometrial tissue samples from women with and without endometriosis were analyzed.
  • immunoblots prepared from eutopic endometrium of women with and without endometriosis were studied for differences in TrkB expression. Elevated levels of TrkB are associated with endometriosis.
  • the present invention provides methods, assays and kits for determining the level of expression of TrkB in various types of biological samples. Quantification of TrkB can be used to diagnose endometriosis as well as to follow the progression or regression of the disease.
  • TrK A, B, and C in gynaecological pathologies such as EAOC This provides novel markers for diagnosis and potential therapeutic targets.
  • Tyrosine kinase receptor A, B and C are expressed in endometriosis associated ovarian cancer specimens.
  • the immunolocalization of tyrosine kinase receptor (types A, B and C) in endometriosis associated ovarian cancer cases is shown.
  • the localization of these receptors in the collected pathological specimens was also determined.
  • a method of diagnosing or predicting endometrial disease comprises: obtaining a sample from a subject; quantitating the level of a tyrosine kinase selected from the group consisting of TrkA, Trk ⁇ , and TrkC expression; and comparing the level determined to a predetermined cut-off value wherein a level higher than the cut-off value is indicative of endometriosis.
  • the endometrial disease is endometriosis or endometriosis associated ovarian cancer (EAOC).
  • the tyrosine kinase is Trk ⁇ .
  • the level of Trk expression may be determined by measuring protein levels or by quantitating Trk ⁇ mRNA.
  • a method of assessing whether a subject is afflicted with endometrial disease comprises comparing the level of expression of Trk in a sample from a subject; and the normal level of expression of Trk in a control sample, wherein a significant difference between the level of expression of Trk in the sample from the subject and the normal level is an indication that the subject is afflicted with endometrial disease.
  • a Trk ⁇ gene product may Trk ⁇ mRNA or cDNA.
  • the invention also provides a method for monitoring the progression or regression of endometrial disease in a subject, said method comprising the steps of detecting a first test amount of Trk ⁇ gene product in a sample from the subject at a first time: detecting a second sample determining a second test amount of the Trk ⁇ gene product in a sample from the subject at a second later time: and comparing the first test amount with the second test amount, wherein an increase in the amount between the first time and the second time indicates progression of endometrial disease and a decrease in the amount between the first time and the second time indicates remission of endometrial disease.
  • the invention further provides a method for predicting a predisposition to endometrial disease in an individual, said method comprising: determining the level of a Trk ⁇ gene product in a sample from the individual and comparing the level of expression of the gene product to a predetermined cut-off value of Trk ⁇ expression, wherein a level of Trk ⁇ greater than the cut-off level of Trk ⁇ is indicative of a predisposition to endometrial disease.
  • a composition for the treatment of endometrial disease comprises an agent that reduces the expression or activity of Trk ⁇ and an acceptable carrier.
  • the agent may be selected from the group consisting of lariat-form RNA, antisense RNA, short temporary RNA (stRNA), small interfering RNA (siRNA), short hairpin RNA (shRNA) micro RNA, aberrant RNA containing mismatches, double stranded RNA (dsRNA) and long deoxyrobonucleotide containing RNA (D-RNA).
  • composition for the modulation of TDAG51 activity may comprise a pharamacological agent such as, a small molecule, a binding peptide, an RNA aptamer or a DNA aptamer or a known drug.
  • a pharamacological agent such as, a small molecule, a binding peptide, an RNA aptamer or a DNA aptamer or a known drug.
  • the invention also provides a method of treating endometrial disease comprising administering a composition as described above.
  • a method of diagnosing endometrial disease comprises: i) obtaining a sample from a subject; ii) quantitating the level of TrkB expression; and iii) comparing the level determined in step M) to a predetermined cut-off value wherein a level higher than the cut-off value is indicative of endometrial disease.
  • the sample is preferably selected from the group consisting of: menstrual fluid, endometrial cells, blood, uterine, cervical, ovarian or vaginal biopsy material, peritoneal fluid and vaginal secretions.
  • the level of TrkB expression may be determined by measuring Trk ⁇ protein levels or TrkB mRNA.
  • another method of assessing whether a subject is afflicted with endometrial disease comprises comparing the level of expression of TrkB in a sample from a subject; and the normal level of expression of TrkB in a control sample, wherein a significant difference between the level of expression of TrkB in the sample from the subject and the normal level is an indication that the subject is afflicted with endometrial disease.
  • the sample typically comprises ectopic endometrial tissue, eutopic endometrial tissue, menstrual fluid, endometrial cells, blood, urine, uterine, cervical, ovarian or vaginal biopsy material, peritoneal fluid and vaginal secretions.
  • the TrkB gene product is TrkB mRNA or cDNA.
  • the TrkB gene product is a TrkB polypeptide or a fragment thereof.
  • the polypeptide or fragment may be detected using an immunoassay.
  • the immunoassay may be a non-competitive immunoassay or a competitive immunoassay. It may be a direct immunoassay or a capture assay.
  • the assay system may be provided as a set of reagents or as a prepared assay kit including complimentary binding reagents bound to a solid surface or in liquid form.
  • Various types of assays for the detection of gene products, whether nucleic acids or proteins, are encompassed within the scope of the invention. This includes dip sticks, adhesive assay strips, etc.
  • the polypeptide or fragment is detected in a tissue sample using immunohistochemical techniques.
  • a method for monitoring the progress or regression of endometrial disease in a subject comprises the steps of detecting a first test amount of TrkB gene product in a sample taken from the subject at a first time: detecting a second test amount of the TrkB gene product in a sample from the subject taken at a second later time: and comparing the first test amount with the second test amount, wherein an increase in the amount between the first time and the second time indicates progression of endometrial disease and a decrease in the amount of a TrkB gene product between the first time and the second time indicates remission of endometrial disease.
  • a method for predicting an increased risk of endometrial disease in an individual comprises determining the level of a TrkB gene product in a sample from the individual and comparing the level of expression of the gene product to a predetermined cut-off value of TrkB expression, wherein a level of TrkB greater than the cut-off level of TrkB is indicative of an increased risk of endometrial disease or early stage endometrial disease.
  • FIGURE 1 shows haematoxylin and eosin and TrkB-stained human endometrium.
  • FIGURE 2 shows TrkB protein expression in human endometrium.
  • FIGURE 3A is a representative photomicrograph of TrkA staining in clear carcinoma cells of a sample taken from a patient with EAOC. Positively stained cells are shown in both high and low magnification;
  • FIGURE 3B is a representative photomicrograph of TrkA staining in ovarian endometriosis sample taken from a patient within study. Positively stained cells are shown in both high and low magnification;
  • FIGURE 4A is a representative photomicrograph of TrkB staining in clear carcinoma cells of a sample taken from a patient with EAOC. Positively stained cells are shown in both high and low magnification;
  • FIGURE 4B is a representative photomicrograph of TrkB staining in ovarian endometriosis sample taken from a patient within study. Positively stained cells are shown in both high and low magnification;
  • FIGURE 5A is a representative photomicrograph of TrKC staining in a low grade serous EAOC
  • FIGURE 5B is a representative photomicrograph of TrKC staining in an ovarian endometriosis sample
  • FIGURE 6 is a series of graphical depictions of staining intensity of TrkA, TrkB and TrkC found in samples taken from A) endometriosis associated ovarian cancer; B) endometriosis; C) ovarian stroma; and D) eutopic endometrium, lmmunohistochemical staining intensity of each individual sample tested was rated as negative, weak, moderate or strong.
  • the present invention provides methods, assay systems, kits, and devices for diagnosing and/or determining the risk of endometriosis and endometriosis associated ovarian cancer (EAOC).
  • EAOC endometriosis and endometriosis associated ovarian cancer
  • the invention also provides methods and assays, including assay kits and devices for determining the progression or regression of the disease by measuring the level of TrkB expression in a sample from a patient.
  • TrkB expression is shown to be increased in women with endometriosis.
  • the increased expression of TrkB in endometriosis as compared with normal endometrium suggests that TrkB is an important diagnostic marker of endometriosis and an indicator of the degree of progression of the disease.
  • TrkB may also be a therapeutic target for the treatment or and/or prevention or progression of endometriosis.
  • FIGURE 1 shows haematoxylin and eosin and TrkB-stained human endometrium.
  • FIGURE 2 shows TrkB protein expression in human endometrium, lmmunoblot demonstrating full-length and truncated TrkB protein expression (145 and 95 kD, respectively) in eutopic endometrium of reproductively aged women with (lanes 1-4) and without (lanes 5 and 7; leiomyomas) endometriosis.
  • the sample in lane 6 was from a woman with menorrhagia but with no evidence of endometriosis.
  • FIGURE 3A is a representative photomicrograph of TrkA staining in clear carcinoma cells of a sample taken from a patient with EAOC. Positively stained cells are shown in both high and low magnification.
  • FIGURE 3B is a representative photomicrograph of TrkA staining in ovarian endometriosis sample taken from a patient within study. Positively stained cells are shown in both high and low magnification.
  • FIGURE 4A is a representative photomicrograph of TrkB staining in clear carcinoma cells of a sample taken from a patient with EAOC. Positively stained cells are shown in both high and low magnification.
  • FIGURE 4B is a representative photomicrograph of TrkB staining in ovarian endometriosis sample taken from a patient within study. Positively stained cells are shown in both high and low magnification.
  • FIGURE 5A is a representative photomicrograph of TrkC staining in a low grade serous EAOC.
  • FIGURE 5B is a representative photomicrograph of TrkC staining in an ovarian endometriosis sample.
  • FIGURE 6 is a series of graphical depictions of staining intensity of
  • TrkA, TrkB and TrkC found in samples taken from A) endometriosis associated ovarian cancer; B) endometriosis; C) ovarian stroma; and D) eutopic endometrium, lmmunohistochemical staining intensity of each individual sample tested was rated as negative, weak, moderate or strong.
  • TrkC receptor was present in all but one of the tissue samples collected in this study. However, in that eutopic endometrium sample there were hyperblastic regions that did stain positive and normal endometrium staining negative.
  • TrkB was localized to epithelial cells and characterized by mainly weak and some moderate cytoplasmic staining.
  • the positive staining patterns in the rest of the samples were generally on a continuum from weakly to strongly cytoplasmic positivity. There was no staining in the negative control specimens.
  • Trk A, B and C expression are evidence of Trk A, B and C expression in endometriosis associated ovarian cancer cases.
  • the tyrosine kinase receptors are expressed differentially across various tissues samples from EAOC cases, with TrkA and TrkB experiencing mainly positive staining patterns in EAOC and endometriosis tissue samples.
  • TrkA and TrkB are generally discriminate in their expression across different tissues samples.
  • the invention provides tools for exploring the interactions present on a molecular level to gain a further understanding of the characteristics and possibly etiology of EAOC. TrkA and TrkB may therefore be used as biomarkers to help identify endometriosis associated ovarian cancers and its process of development and propagation.
  • TrkB is expressed in epithelial cells of more aggressive EAOC compared with less aggressive tumours or benign tissues.
  • the present invention provides methods, assays and kits that measure
  • TrkA, B and C gene products are used to encompass both polypeptides and nucleic acids encoding TrkA, B and C.
  • Gene products useful in the invention include mRNA, the cDNA derived therefrom, a polypeptide or a portion of a polypeptide.
  • the present invention provides methods for providing a diagnosis of endometrial disease as well as methods for following the progression and/or regression of endometrial disease.
  • the methods of the invention can also be used to aid a physician in the management of endometrial disease by providing an indication of the aggressiveness of the disease.
  • the identification of tyrosine kinase markers provides for novel therapeutic targets and methodologies.
  • the invention therefore also includes methods of treatment of endometriosis and EAOC.
  • inhibitors of TrkB expression or activity may be used.
  • the methods of the invention can also be used to predict whether a given woman is likely to develop endometrial disease, such as endometriosis for EAOC.
  • One method of determining the susceptibility of a patient comprises the steps of (i) obtaining a sample containing nucleic acids and/or polypeptides, from endometrial cells of a patient; and (ii) determining whether the sample contains a level of TrkA, B or C, preferably TrkB, nucleic acid or protein associated with endometrial disease.
  • the level in the test sample is compared to a baseline level obtained by analysis of multiple samples from women without endometriosis.
  • a method of diagnosing endometrial disease in a human patient comprising the steps of (i) obtaining a sample containing nucleic acid and/or protein from endometrial cells of a patient; and (ii) determining whether the sample contains a level of TrkB nucleic acid or protein associated with endometrial disease.
  • the level of TrkA, B or C which is determined to be indicative of endometrial disease may be based on a predetermined baseline value.
  • the readout may be a comparison with a normal sample. For example, a reading at least 1 1 /2 fold higher, or 3-fold higher than a control sample may be determined to be indicative of endometrial disease.
  • TrkB as a marker for endometrial disease may in itself provide a method for diagnosis or prognosis. Alternatively, the method may be used as an adjunct to known diagnostic and prognostic methods such as histopathological examination of biopsy tissue, or laparoscopy.
  • the level of the TrkB in a sample may also be used to determine how to manage the patient. For example, very high levels may indicate extensive disease and suggest that aggressive therapeutic options be pursued. On the other hand, if the level is moderately elevated, less invasive therapeutic options may be considered more appropriate.
  • TrkB levels appear to be an early marker of endometriosis and changes in the expression of TrkB may precede other signs of endometriosis.
  • An elevated level of TrkBin a sample from a patient that has no visible or easily detected signs of endometriosis may indicate that the patient is in the early stages of developing endometriosis. Alternatively it may indicate that the patient may later develop endometriosis, or that the patient is particularly susceptible to risk factors for developing endometriosis. High levels of expression may also be indicative of EAOC.
  • the patient is a human patient although endometrial disease may occur in other mammals.
  • the sample for analysis is preferably selected from the group consisting of endometrial tissue, menstrual fluid, endometrial cells, blood, uterine, cervical, ovarian or vaginal biopsy material, peritoneal fluid and vaginal secretions.
  • Endometrial tissue may be obtained by surgical excision, laparoscopy and biopsy, and image-guided biopsy.
  • biopsy material is the richest source of ectopic endometrial cells
  • Cells derived from endometriotic lesions may be found in small numbers in menstrual fluid, peritoneal fluid, urine, vaginal secretions, and blood.
  • the sample containing the nucleic acid or polypeptide from the patient is, or was derived directly from, a cell of the patient, analysis of a sample indirectly derived from a patient, such as a cell grown in culture, is also included within the invention.
  • the methods of the invention may be used for presymptomatic screening of a patient who has been identified as being in a risk group for endometrial disease, such as a woman having a family history of endometrial disease.
  • the methods may also be used as a primary screen for patients exhibiting symptoms which may or may not be related to endometrial disease, such as difficulty getting pregnant, pelvic pain, painful periods or pain during intercourse.
  • the test sample can be obtained from various sources if a fluid source such as blood, vaginal secretions, or urine is used as the test sample. It is preferable to enrich the sample for endometriosis-derived tissue or cells. Enrichment for endometrial cells may be achieved using, for example, cell- sorting methods such as fluorescent activated cell sorting (FACS) using an endometriosis-selective antibody.
  • FACS fluorescent activated cell sorting
  • TrkA a level of TrkA, B or C, in particular
  • nucleic acid or protein associated with endometrial disease may be determined to be a level of TrkBnucleic acid or protein that is undetectable in normal tissue.
  • a sample known to contain TrkBnucleic acid or protein for example a previously tested malignant endometrial disease biopsy sample or a sample of a cell line, may be used as a reference sample.
  • Another of a single cell or a population of cells reference sample may be a previously tested normal biopsy sample.
  • the methods of the invention may utilize various types of analysis.
  • the method allows for the determination of TrkB xpression in individual cells.
  • the results of the method may be expressed, for example, as a presence of TrkB in any of the cells tested, absence of TrkB in all of the cells tested, presence of TrkB in all of the cells tested, proportion of the cells tested in which the presence of TrkB was detected, or a numerical average of the quantified level of TrkB across all the cells tested, or across those cells in which TrkB expression was detected.
  • TrkB expression is determined by measuring the presence or absence or amount of a TrkB gene product.
  • Preferred gene products that are useful for determining TrkB expression are TrkB mRNA and TrkB polypeptide or fragments thereof.
  • the level of TrkB nucleic acid in particular mRNA or the cDNA derived therefrom, is measured.
  • the level of TrkB is measured by contacting a sample containing nucleic acid with a nucleic acid which hybridizes selectively to the TrkB nucleic acid.
  • selectively hybridizing is used herein to indicate that the nucleic acid has sufficient nucleotide sequence similarity with the said human nucleic acid that it can hybridize under moderately or highly stringent conditions, and does not hybridize to other nucleic acids under the same conditions.
  • nucleic acid hybridization depends on factors such as length of nucleic acid over which hybridization occurs, degree of identity of the hybridizing sequences and on factors such as temperature, ionic strength and CG or AT content of the sequence.
  • any nucleic acid which is capable of selectively hybridizing is useful in the practice of the invention.
  • Typical moderately or highly stringent hybridization conditions which lead to selective hybridization are known in the art, for example those described in Molecular Cloning, a laboratory manual, 2nd edition, Sambrook et al (eds), Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., USA, incorporated herein by reference.
  • the mRNA gene product, or the cDNA derived therefrom can also be determined by quantitative PCR, in situ hybridization with antisense RNA or through the use of a DNA microarray. Chromogenic assays may be used.
  • the level of TrkB polypeptide is measured.
  • Various methods such as direct or indirect immunoassays may be used to detect TrkB polypeptides.
  • an ELISA, RIA or other conventional assays can be used to detect TrkB in fluid samples.
  • Immuno-histochemistry can be used to detect and quantitate TrkBin tissue.
  • Direct or indirect assays involving labeled antibodies or ligands are useful in the practice of the invention.
  • kits for performing the methods and assays of the invention may comprise one or more detection reagents which bind to TrkBnucleic acid gene product.
  • the kit may include at least one antibody capable of binding to a TrkB olypeptide and a detecting agent.
  • Example 1 Study subjects and tissue sample collection
  • Endometrial biopsies were obtained from women between the ages of
  • Example 2 Routine Histology and lmmunohistochemistry [0074] 5 ⁇ m paraffin sections were stained with haematoxylin and eosin to identify the reproductive cycle stage (proliferative or secretory) of each patient at the time of surgery. While blinded to patient diagnosis (i.e. endometriosis or endometriosis-free), immunohistochemical staining was undertaken to identify the presence of TrkB Sections were deparaffinized using xylene, brought to water through graded ethanol solutions, rinsed in phosphate buffered saline (PBS), then incubated in a 1 % solution of H 2 O 2 in methanol for 30' to inhibit endogenous peroxidases.
  • PBS phosphate buffered saline
  • Non-specific binding was blocked for one hour using 1.5% normal goat serum (Vectastain ABC kit, Vector Laboratories Inc., Burlingame, CA, U.S.A.) in PBS with 1 % bovine serum albumin. Sections were then incubated overnight at 4°C with 2 ⁇ g/mL polyclonal rabbit TrkBantibody (sc-8316; Santa Cruz Biotechnology Inc., Santa Cruz, CA. U.S.A.) in PBS with 1 % BSA. Negative controls were incubated with non-immune rabbit lgd serum. Sections were washed three times with PBS followed by a 2 h incubation in biotinylated secondary antibody for two hours at room temperature.
  • Vectastain ABC kit Vector Laboratories Inc., Burlingame, CA, U.S.A.
  • the slides were washed and incubated with Vectastain ABC Reagent (avidin DH and biotinylated horseradish peroxidase H; Vector Laboratories Inc.) for two hours at room temperature.
  • Vectastain ABC Reagent avidin DH and biotinylated horseradish peroxidase H; Vector Laboratories Inc.
  • DAB Diaminobenzidine tetra hydrochloride
  • Sections were dehydrated through graded ethanol solutions, cleared in xylene, and mounted with Permount for bright-field microscopy. Images were acquired digitally using an Olympus microscope coupled to an image analysis system (Image Pro Plus, Media Cybernetics, Silver Spring, U.S.A).
  • Table I Mean H-scores for TrkB protein expression in eutopic endometrium over the menstrual cycle in women with and without endometriosis.
  • Proteins were extracted from tissues by homogenization in RIPA buffer containing 1 % Triton-X, 3.5 mM SDS, 0.2 M NaCI, 0.2 M Tris-HCI, 0.01 M deoxycholic acid sodium salt (Sigma Aldrich, Oakville, Ont., Canada) and Complete Mini protease inhibitor (1 tablet per 10 ml_; Roche Diagnostics, Laval, Que., Canada). Homogenates were centrifuged at 200O x g for 15 min at 4 ° C.
  • This antibody recognized both full-length (145 kDa) and truncated (95 kDa) forms of the receptor.
  • membranes were washed with PBS/Tween and incubated with horseradish peroxidase conjugated secondary antibody (1 :5000 anti-rabbit IgG; Amersham Biosciences Inc., Piscataway, NJ, U.S.A.) in blocking buffer for one hour.
  • Enhanced chemiluminescent detection ECL; Amersham Biosciences was used to visualize the protein bands. Band density was assessed and normalized to ⁇ -actin (AbCam Inc., Cambridge, MA, U.S.A.) used as a loading control.
  • TrkB protein was abundantly expressed in endometrial biopsies of women with endometriosis compared to samples from a reference population without endometriosis (Fig. 2).
  • TrkB protein levels in samples from women with menorrhagia with no evidence endometriosis were comparable to those of women with the disease (Fig. 2).
  • Example 5 Expression of TrkA, TrkB and TrkC in various tissues.
  • Study subjects A total of 75 women were identified through a search of pathology records (Department of Pathology at McMaster University Medical Center) for all cases of EAOC. Chart reviews and tissue sample retrievals were approved by the Research Ethics Board, McMaster University. All procedures were conducted in accordance with McMaster University Medical Centre guidelines. Only those cases with EOAC, endometriosis, ovarian stroma and endometrium samples were used for further analysis. Archived samples of formalin-fixed, paraffin embedded blocks from 15 women with EAOC.
  • Immunohistochemistry Paraffin blocks ovarian tumor, ovarian endometriosis, eutopic endometrium, and normal ovarian stroma for each subject were retrieved and 5 ⁇ M thick sections were prepared for routine and immunohistochemistry. One section from each block was stained with haematoxylin and eosin and reviewed by a pathologist blind to previous diagnosis. Only sections containing ovarian tumor, ovarian endometriosis, eutopic endometrium, and normal ovarian stroma were used for further immunohistochemical study.
  • Sections were then incubated overnight at 4°C with primary rabbit polyclonal antibody diluted in PBS with 1 % BSA at the following dilutions: TYkB (sc-8316; recognizes both the full-length and truncated receptor isoforms) Tissue sections were incubated with a polyclonal rabbit anti-human TrkA Ab (1 :250), TrkB Ab (1 :250), or TrkC Ab (1 :250 (Santa Cruz Biotechnology Inc., Santa Cruz, CA. U.S.A.). Negative controls were incubated with PBS/BSA solution with the primary antibody omitted. Sections were washed three times with PBS followed by incubation with biotinylated secondary antibody at room temperature for two hours.
  • TYkB sc-8316; recognizes both the full-length and truncated receptor isoforms
  • the slides were then washed and incubated with Vectastain ABC Reagent (avidin DH and biotinylated horseradish peroxidase H) for two hours at room temperature.
  • Vectastain ABC Reagent avidin DH and biotinylated horseradish peroxidase H
  • 0.25 mg/mL diaminobenzidine tetrahydrochloride (DAB; Sigma-Aldrich) was used as the chromagen for colour development, followed by counterstaining with Harris hematoxylin (Sigma-Aldrich) for one minute. Acid alcohol and Scott's tap water were used as done previously for hematoxylin and eosin staining. Sections were finally dehydrated through graded ethanol solutions, cleared in xylene, and mounted for bright-field microscopy.
  • the calculated mean age was 51 years ⁇ 3.42, ranging from 31 to 76 years of age.
  • the average size of the tumor was 10.5 ⁇ 1.84 cm with a range from 0.5 to 27 cm. Metastasis of the tumor occurred in 9 of the final 14 cases. Four of the cases were bilateral.

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Abstract

La présente invention concerne des procédés, des dosages et des kits destinés au diagnostic et au traitement de l'endométriose et du cancer des ovaires associé à une endométriose. L'invention repose sur la détection de niveaux élevés d'expression du récepteur de kinase lié à la tropomyosine du facteur de croissance du tissu nerveux (récepteurs de kinase tyrosine TrkA, Trkβ et TrkC) dans des échantillons de tissu et des fluides corporels, par détermination des niveaux de protéine ou des niveaux mRNA de Trk.
PCT/CA2007/002114 2006-11-24 2007-11-23 Procédé de diagnostic permettant de détecter l'endométriose Ceased WO2008061370A1 (fr)

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Publication number Priority date Publication date Assignee Title
WO2015017533A1 (fr) * 2013-07-30 2015-02-05 Blueprint Medicines Corporation Fusions de ntrk2

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WO2007049041A1 (fr) * 2005-10-28 2007-05-03 Astrazeneca Ab Derives 4-(3-aminopyrazole)pyrimidine et leur utilisation en tant qu’inhibiteurs de tyrosine kinases dans le cadre d’un traitement anticancereux

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Publication number Priority date Publication date Assignee Title
WO2007049041A1 (fr) * 2005-10-28 2007-05-03 Astrazeneca Ab Derives 4-(3-aminopyrazole)pyrimidine et leur utilisation en tant qu’inhibiteurs de tyrosine kinases dans le cadre d’un traitement anticancereux

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CAMPOS X. ET AL.: "Nerve growth factor and its high-affinity receptor trkA participate in the control of vascular endothelial growth factor expression in epithelial ovarian cancer", GYNECOLOGIC ONCOLOGY, vol. 104, no. 1, January 2007 (2007-01-01), pages 168 - 175, XP005734228, DOI: doi:10.1016/j.ygyno.2006.07.007 *
DAVIDSON B. ET AL.: "Expression levels of the nerve growth factor receptors TrkA and p75 in effusions and solid tumors of serous ovarian carcinoma patients", CLINICAL CANCER RESEARCH, vol. 7, no. 11, November 2001 (2001-11-01), pages 3457 - 3464 *
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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2015017533A1 (fr) * 2013-07-30 2015-02-05 Blueprint Medicines Corporation Fusions de ntrk2
US10407509B2 (en) 2013-07-30 2019-09-10 Blueprint Medicines Corporation NTRK2 fusions
EP3628749A1 (fr) * 2013-07-30 2020-04-01 Blueprint Medicines Corporation Fusions de ntrk2

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