WO2008061370A1

WO2008061370A1 - Diagnostic method to detect endometriosis

Info

Publication number: WO2008061370A1
Application number: PCT/CA2007/002114
Authority: WO
Inventors: Warren Foster
Original assignee: McMaster University
Current assignee: McMaster University
Priority date: 2006-11-24
Filing date: 2007-11-23
Publication date: 2008-05-29
Anticipated expiration: 2009-05-24

Abstract

The invention provides methods, assays and kits for the diagnosis and treatment of endometriosis and endometriosis associated ovarian cancer. The invention is based on the detection of elevated levels of nerve growth factor (NGF) tropomyosin-related kinase receptor (tyrosine kinase receptors TrkA, Trkβ and TrkC) expression in tissue samples and bodily fluids by determining protein levels or mRNA levels of Trk.

Description

Diagnosis of endometriosis by tropomyosm-related kinase receptor expression

FIELD OF INVENTION

[oooi] The present invention relates to methods and compositions for detecting and monitoring endometrial disease, such as endometriosis and endometriosis associated ovarian cancer.

BACKGROUND OF THE INVENTION

[0002] 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. However, the critical event(s) or biochemical change(s) that ultimately leads to establishment of endometriosis remains unknown.

[0003] Several distinct lines of evidence suggest that 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. For example, dysregulation of the endometrial growth inhibitory factor, interleukin-6 (IL- 6), and its soluble receptor ( I L-6s R) has been shown in ectopic endometrium. Also, aromatase is expressed in ectopic endometrium from women with endometriosis but not in the endometrium from women without disease. This suggests that there may be a mechanism through which the ectopic endometrium can synthesize its own estrogen to promote implant survival. In women with endometriosis, menstruated endometrial cells and tissue fragments resist degradation and, compared to women without endometriosis, rates of apoptosis are also lower in the eutopic endometrium. The mechanisms regulating apoptosis in the endometrium are not known. Thus, the molecular mechanisms involved in the development and progression of endometriosis remain to be elucidated and there are no easy ways to diagnose endometriosis. Current methods involve physical examination, laparoscopy and biopsy and even those methods have their limitations. There is an unmet need for a greater understanding of the mechanisms involved that can lead to better methods of diagnosis.

[0004] A related disorder, endometriosis associated ovarian cancer (EAOC), often appears as either a low-grade tumour of endometrioid cell type or as a clear cell tumour in young women. To date, EAOC remains a rare disease with unknown etiology. Endometriosis has been reported to coexist with approximately 10%-15% of ovarian cancer cases. Endometriosis and EAOC share several common features including the propensity of progressive and invasive, estrogen-dependent growth, reoccurrence as well as the tendency to metastasize. It is thought that endometriosis on the ovary may precede the development of EAOC, suggesting a shared pathophysiology.

[0005] 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.

[0006] Tropomyosin-related kinase receptors A, B and C are neutrophin receptors that regulate the proliferation, differentiation and programmed cell death of neuronal cells. There is substantial evidence in the literature that shows that these receptors play a critical role in both the central and peripheral nervous system. Nevertheless, evidence of Trk activity in areas outside of the nervous system has also been put forth. Recent data also reveals involvement of these receptors in tumour development. TrkA expression in pancreatic cancer has been shown to act as a marker of tumour aggressiveness and invasiveness. There is evidence also suggesting that 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. 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 have been localized to the granulosa, cumulus and theca interna cells of preovluatory follicles and TrkB receptor was demonstrated in mammalian oocytes. 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.

[0007] Despite considerable research, a genetic marker that is useful in reliably predicting and/or diagnosing endometrial disease has not been identified.

SUMMARY OF THE INVENTION

[0008] The present invention provides methods for the detection, prediction, and staging of endometrial diseases. Therapeutic compositions and methods are also provided. As used herein, the term "endometrial disease" refers to any disorder associated with aberrant proliferation or localization of endometrial cells. This includes endometriosis and endometriosis associated ovarian cancer (EAOC). [0009] The tyrosine receptor kinases (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. Of the tyrosine receptor kinases, TrkB is a neurotrophic receptor best known for its role in the nervous system where it regulates cellular proliferation, differentiation and survival. Moreover, 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.

[ooio] 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. In addition, 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.

[ooi l] The present invention also demonstrates for the first time expression of

TrK A, B, and C in gynaecological pathologies such as EAOC. This provides novel markers for diagnosis and potential therapeutic targets.

[ooi2] 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.

[ooi3] In one aspect of the invention, a method of diagnosing or predicting endometrial disease is provided. The method comprises: obtaining a sample from a subject; quantitating the level of a tyrosine kinase selected from the group consisting of TrkA, Trkβ, 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.

[ooi4] In preferred embodiments, the endometrial disease is endometriosis or endometriosis associated ovarian cancer (EAOC).

[ooi5] In a further preferred embodiment, the tyrosine kinase is Trkβ. The level of Trk expression may be determined by measuring protein levels or by quantitating Trkβ mRNA.

[0016] In another aspect, a method of assessing whether a subject is afflicted with endometrial disease is provided. The method 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 Trkβ gene product may Trkβ mRNA or cDNA.

[ooi7] 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 Trkβ gene product in a sample from the subject at a first time: detecting a second sample determining a second test amount of the Trkβ 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. [0018] The invention further providesa method for predicting a predisposition to endometrial disease in an individual, said method comprising: determining the level of a Trkβ 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 Trkβ expression, wherein a level of Trkβ greater than the cut-off level of Trkβ is indicative of a predisposition to endometrial disease.

In another aspect of the invention, a composition for the treatment of endometrial disease is provided. The composition comprises an agent that reduces the expression or activity of Trkβ 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). The 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. The invention also provides a method of treating endometrial disease comprising administering a composition as described above.

[0019] In one preferred aspect of the invention a method of diagnosing endometrial disease is provided. The method 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.

[0020] 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.

[0021] The level of TrkB expression may be determined by measuring Trkβ protein levels or TrkB mRNA.

[0022] In another aspect of the invention, another method of assessing whether a subject is afflicted with endometrial disease is provided. The method 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.

[0023] 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.

[0024] In one preferred embodiment, the TrkB gene product is TrkB mRNA or cDNA.

[0025] In another embodiment, 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.

[0026] In another embodiment, the polypeptide or fragment is detected in a tissue sample using immunohistochemical techniques.

[0027] In another aspect of the invention, a method for monitoring the progress or regression of endometrial disease in a subject is provided. The method 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. [0028] In a further aspect of the invention, a method for predicting an increased risk of endometrial disease in an individual is provided. The method 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.

BRIEF DESCRIPTION OF THE DRAWINGS

[0029] These and other features of the invention will become more apparent from the following description in which reference is made to the appended drawings wherein:

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.

DETAILED DESCRIPTION

[0030] 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). 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.

[0031] The diagnosis of endometriosis is generally considered uncertain until proven by laparoscopy. Even so, laparoscopy is not always, conclusive. Skill and familiarity with endometriosis are important as it has many different appearances and may be missed by the physician. If endometriosis is seen at laparoscopy, a biopsy is usually taken to confirm the diagnosis. However, a biopsy is an intrusive procedure and there has been an unmet need for less aggressive procedures. Ultrasound, computerized tomography (CT scan) or magnetic resonance imaging (MRI) can be used to get more information about the extent of endometriosis in some cases. These techniques however cannot be used to make a definite diagnosis of endometriosis. Thus, present screening methods are not entirely satisfactory and there is no easy way to confirm the presence of endometriosis. In one aspect, the present invention addresses the need for non-invasive tests to diagnose and stage endometriosis.

[0032] In the present invention 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.

[0033] Referring now to the figures, FIGURE 1 shows haematoxylin and eosin and TrkB-stained human endometrium. H&E stained eutopic endometrium from reporoductively-aged women in the A) proliferative; B) secretory phases of the menstrual cycle; C) Trkβ staining was observed throughout epithelial and stromal cells of eutopic endometrium in both women with and without endometriosis regardless of cycle stage. Tissue from a woman with endometriosis in the secretory stage is also shown; and D) negative control using non-immune rabbit serum instead of Trkβ antibody. These results indicate that TrkB can be differentially detected in tissue samples.

[0034] 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. These results indicate that elevated expression of TrkB is associated with endometriosis.

[0035] Tyrosine kinase receptor (types A, B and C) staining in endometriosis associated ovarian cancer (EAOC) cases was also analyzed. Additionally, localization of these receptors in the collected pathological specimens was determined.

[0036] The results of these studies are shown in Figures 3-6. [0037] 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.

[0038] 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.

[0039] 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.

[0040] 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.

[0041] FIGURE 5A is a representative photomicrograph of TrkC staining in a low grade serous EAOC.

[0042] FIGURE 5B is a representative photomicrograph of TrkC staining in an ovarian endometriosis sample.

[0043] 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.

[0044] It was found that the 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.

[0045] In terms of staining patterns, 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.

[0046] There is 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.

[0047] The results suggest that 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.

[0048] When cancer type was examined in the EAOC cases, it was found that

TrkB is expressed in epithelial cells of more aggressive EAOC compared with less aggressive tumours or benign tissues.

[0049] The present invention provides methods, assays and kits that measure

TrkA, B and C gene products. As used herein, the term "gene product" is 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.

[0050] In addition, 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. For example, inhibitors of TrkB expression or activity may be used. [0051] 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. In a preferred embodiment, the level in the test sample is compared to a baseline level obtained by analysis of multiple samples from women without endometriosis.

[0052] In another aspect of the invention, there is provided a method of diagnosing endometrial disease in a human patient, said method 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.

[0053] The level of TrkA, B or C which is determined to be indicative of endometrial disease may be based on a predetermined baseline value. Alternatively, 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.

[0054] The use of 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.

[0055] 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.

[0056] 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.

[0057] It is preferred that 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.

[0058] While biopsy material is the richest source of ectopic endometrial cells, it is desirable to obtain test samples using non-invasive procedures. Cells derived from endometriotic lesions may be found in small numbers in menstrual fluid, peritoneal fluid, urine, vaginal secretions, and blood. Although it is preferred that 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.

[0059] 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.

[0060] As discussed above, 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.

[0061] In the methods of the invention, a level of TrkA, B or C, in particular

TYkB, 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.

[0062] It will be appreciated that 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.

[0063] It will be appreciated that reference to determining the level of TrkB expression includes the meaning of determining whether TrkB expression may be detected or not. 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.

[0064] In one preferred embodiment of the invention, 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. The term "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. As is well known in the art, the stringency of 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. Thus, any nucleic acid which is capable of selectively hybridizing is useful in the practice of the invention.

[0065] 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.

[0066] 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.

[0067] In another preferred embodiment, the level of TrkB polypeptide is measured. Various methods, such as direct or indirect immunoassays may be used to detect TrkB polypeptides. For example, 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.

[0068] The present invention also includes kits for performing the methods and assays of the invention. In one embodiment, the kit may comprise one or more detection reagents which bind to TrkBnucleic acid gene product. In another embodiment, the kit may include at least one antibody capable of binding to a TrkB olypeptide and a detecting agent.

[0069] One or more currently preferred embodiments have been described by way of example. It will be apparent to persons skilled in the art that a number of variations and modifications can be made without departing from the scope of the invention as defined in the claims. [0070] The above disclosure generally describes the present invention. It is believed that one of ordinary skill in the art can, using the preceding description, make and use the compositions and practice the methods of the present invention. A more complete understanding can be obtained by reference to the following specific examples. These examples are described solely to illustrate preferred embodiments of the present invention and are not intended to limit the scope of the invention. Changes in form and substitution of equivalents are contemplated as circumstances may suggest or render expedient. Other generic configurations will be apparent to one skilled in the art. All journal articles and other documents such as patents or patent applications referred to herein are hereby incorporated by reference.

EXAMPLES

[0071] Although specific terms have been used in these examples, such terms are intended in a descriptive sense and not for purposes of limitation. Methods of molecular biology, biochemistry and chemistry referred to but not explicitly described in the disclosure and these examples are reported in the scientific literature and are well known to those skilled in the art.

Example 1. Study subjects and tissue sample collection

[0072] Archived samples of formalin-fixed, paraffin-embedded endometrium from 17 female patients of reproductive age (42 ± 2.5 years) were obtained from the Department of Pathology at McMaster University Medical Centre. Seven of these patients had surgically confirmed endometriosis.

[0073] Endometrial biopsies were obtained from women between the ages of

18 and 45 undergoing benign gynecological surgery at McMaster University Medical Centre. All women were cycling normally and had not received hormonal therapy for at least one month prior to surgery. Informed consent was obtained prior to surgery and all procedures were conducted in accordance with McMaster University Research Ethics Board approval.

Example 2. Routine Histology and lmmunohistochemistry [0074] 5 μm 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 H₂O₂ in methanol for 30' to inhibit endogenous peroxidases. 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 μg/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. 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. Diaminobenzidine tetra hydrochloride (DAB) was used as the chromagen for colour development, followed by counterstaining with Carazzi's hematoxylin. 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).

[0075] To quantify the immunohistochemical staining for TrkB H-scores were determined for each section by one investigator who was blinded to the endometriosis diagnosis of each patient. Approximately 1000 glandular cells were counted per section using an orthogonal gridmask placed over images captured at 40x magnification using Image Pro Plus. The following equation was used to calculate the H-scores: H = Σ P/ (/ + 1 ), where / is the staining intensity of the cells (with 0, 1 , 2 and 3 representing zero, weak, moderate and strong staining, respectively) and P/ is the percentage of cells stained at each intensity (0 - 100%).

[0076] The results of immunohistochemical localization of TrkB expression in archived paraffin embedded endometrial tissue samples from women with and without endometriosis are shown in Figure 1. Endometrial samples from both the proliferative and secretory phases were evaluated (Fig. 1A and 1 B). TrkB protein expression was evident in both cycle phases of women with and without endometriosis (Fig. 1C), with cytoplasmic staining occurring in both epithelial and stromal cells of the endometrial tissue (Fig. 1 D). While most glands had staining in all cells, some tissues had glands in which 50% or fewer cells demonstrated TrkB staining. Although there was a trend for increased numbers of cells to stain positive for Trkβ in the endometrium of women with endometriosis in the secretory phase, no significant difference in the H-score was found as shown in Table I below.

Table I. Mean H-scores for TrkB protein expression in eutopic endometrium over the menstrual cycle in women with and without endometriosis.

Cycle Stage N Endometriosis H-Score

Dx

Proliferative 4 No 1.3 ± 0.1

Proliferative 4 Yes 1.2 ± 0.1

Secretory 6 No 1.4 ± 0.1

Secretory 3 Yes 1.6 ± 0.3

^*Note: no significant difference was observed upon comparison of H-score values for cycle stage (P = 0.18), endometriosis diagnosis (P = 0.56), or for interaction between stage and diagnosis (P = 0.29) by 2-way ANOVA. The power of each test was significantly below the desired power of 0.8 due to low N values.

Example 3. Tissue sample preparation and Western blotting [0077] Endometrial biopsy samples from women with endometriosis (n = 14;

39.0 ± 1.8 years) and women without endometriosis (n = 19; 40.5 ± 0.9 years) were immediately frozen and stored at -85°C until use. 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. The supernatants were collected and protein concentration was determined using Bio-Rad protein assay dye reagent (Bio-Rad Laboratories, Hercules, CA, U.S.A.) and the Bradford method (37). Proteins (15 μg) were electrophoresed by 10% SDS-PAGE under reducing conditions and transferred onto nitrocellulose membranes. Nonspecific binding was blocked by incubation with phosphate-buffered saline (PBS) containing 0.5% Tween-20 with 5% nonfat powdered milk overnight at 4⁰C. Membranes were incubated for 1 hour with Trkβ polyclonal antibody (sc-8316; Santa Cruz Biotechnology Inc.) diluted to 1 μg/mL in blocking buffer. This antibody recognized both full-length (145 kDa) and truncated (95 kDa) forms of the receptor. Following incubation with the primary antibody, 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 β-actin (AbCam Inc., Cambridge, MA, U.S.A.) used as a loading control.

[0078] The results for endometrial samples collected from women between the ages of 18 and 45 undergoing hysterectomy for benign gynecological reasons are shown in Figure 2. Using Western blot techniques, TrkB protein was abundantly expressed in endometrial biopsies of women with endometriosis compared to samples from a reference population without endometriosis (Fig. 2). Interestingly, 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 4. Statistical analyses

[0079] Summary statistics and group comparisons were made by 2-way analysis of variance (ANOVA) using SigmaStat (Systat Software, Inc., Richmond, CA, U.S.A.). A P value of < 0.05 was considered significant.

Example 5. Expression of TrkA, TrkB and TrkC in various tissues. [0080] 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.

[0081] Immunohistochemistry: Paraffin blocks ovarian tumor, ovarian endometriosis, eutopic endometrium, and normal ovarian stroma for each subject were retrieved and 5 μM 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. Five micron sections were deparaffinized using xylene, brought to water through graded ethanol solutions, rinsed in 1x phosphate buffered saline (PBS), then incubated in a 3.5% solution of H₂O₂ in methanol for 30 minutes to inhibit the activity of endogenous peroxidases. 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 (Sigma-Aldrich). 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. The slides were then washed and incubated with Vectastain ABC Reagent (avidin DH and biotinylated horseradish peroxidase H) for two hours at room temperature. 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. Images were acquired as performed previously for hematoxylin and eosin staining. The intensity of staining was also determined for each sample. [0082] Statistical methods: Descriptive data are presented as the mean ± standard error of the mean (SEM). All statistical procedures were performed using SigmaStat for Windows (Sigma).

[0083] Of the patients studied, 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.

[0084] The results indicated that tyrosine kinase receptors, type A, B and C, are indeed expressed in the EAOC samples collected for the study with positive staining in 8/1 1 (73%), 7/12 (59%) and 13/13 (100%) cases, respectively. With the exception of TrkC, the receptors were generally not present in ovarian stroma and eutopic endometrium tissue samples. Therefore, with regards to TrkA and TrkB, there were differing staining intensities across the various tissue samples. These differential expressions in pathological specimens of EAOC suggest that neurotrophic factors and their cognate receptors may be important in the pathophysiology of EAOC and endometriosis.

Claims

WHAT IS CLAIMED IS:

1. A method of diagnosing or predicting endometrial disease comprising:

(a) obtaining a sample from a subject;

(b) quantitating the level of a tyrosine kinase selected from the group consisting of TrkA, Trkβ, and TrkC expression; and

(c) comparing the level determined in step ii) to a predetermined cut-off value wherein a level higher than the cut-off value is indicative of endometriosis.

2. A method according to claim 1 , wherein the endometrial disease is endometriosis.

3. A method according to claim 1 , wherein the endometrial disease is endometriosis associated ovarian cancer (EAOC).

4. A method according to claim 1 wherein the tyrosine kinase is TrkB.

5. A method according to claim 1 , wherein the sample is selected from the group consisting of: menstrual fluid, endometrial cells, blood, uterine, cervical, ovarian or vaginal biopsy material, peritoneal fluid and vaginal secretions.

6. A method according to claim 4, wherein the level of TrkB expression is determined by measuring protein levels.

7. A method according to claim 4, wherein TrkB expression is measured by quantitating TrkB mRNA.

8. A method of assessing whether a subject is afflicted with endometrial disease, the method comprising comparing:

(a) the level of expression of TrkB in a sample from a subject; and (b) 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.

9. The method of claim 8 wherein the sample comprises ectopic endometrial tissue, eutopic endometrial tissue, menstrual fluid, endometrial cells, blood, uterine, cervical, ovarian or vaginal biopsy material, peritoneal fluid and vaginal secretions.

10. The method of claim 8, wherein the TrkB gene product is TrkB mRNA or cDNA.

11. A method of claim 8, wherein the TrkB gene product is a TrkB polypeptide.

12. The method of claim 1 1 , wherein the polypeptide is detected using an immunoassay.

13. The method of claim 12 wherein the immunoassay is a non-competitive immunoassay.

14. The method of claim 12, wherein the immunoassay is a competitive immunoassay.

15. 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 Trkβ gene product in a sample from the subject at a first time: detecting a second sample determining a second test amount of the TrkB 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.

16. A method for predicting a predisposition to endometrial disease in an individual, said method comprising: 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 a predisposition to endometrial disease.

17. A composition for the treatment of endometrial disease, said composition comprising an agent that reduces the expression or activity of TrkB and an acceptable carrier.

18. A composition according to claim 17, wherein the agent is 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).

19. A composition for the modulation of TrkA or TrkB expression or activity, said composition comprising a pharamacological agent.

20. A composition according to claim 19 wherein the pharmacological agent is a small molecule, a binding peptide, an RNA aptamer or a DNA aptamer.

21. A composition according to claim 18 further comprising a known drug.

22. A method of treating endometrial disease comprising administering a composition as defined in claim 17.