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WO2013023994A1 - Nouveau biomarqueur pour la classification de tumeurs ovariennes - Google Patents

Nouveau biomarqueur pour la classification de tumeurs ovariennes Download PDF

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Publication number
WO2013023994A1
WO2013023994A1 PCT/EP2012/065610 EP2012065610W WO2013023994A1 WO 2013023994 A1 WO2013023994 A1 WO 2013023994A1 EP 2012065610 W EP2012065610 W EP 2012065610W WO 2013023994 A1 WO2013023994 A1 WO 2013023994A1
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Prior art keywords
cystatin
subject
ovarian
fragments
malignant
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Piet Moerman
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Pronota NV
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Pronota NV
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57407—Specifically defined cancers
    • G01N33/57449—Specifically defined cancers of ovaries
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/46—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans from vertebrates
    • G01N2333/47—Assays involving proteins of known structure or function as defined in the subgroups
    • G01N2333/4701—Details
    • G01N2333/4725—Mucins, e.g. human intestinal mucin
    • 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
    • G01N2333/81—Protease inhibitors
    • G01N2333/8107—Endopeptidase (E.C. 3.4.21-99) inhibitors
    • G01N2333/8139—Cysteine protease (E.C. 3.4.22) inhibitors, e.g. cystatin
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/50—Determining the risk of developing a disease
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/52—Predicting or monitoring the response to treatment, e.g. for selection of therapy based on assay results in personalised medicine; Prognosis

Definitions

  • the invention relates to protein- and/or peptide-based biomarkers useful for classifying ovarian tumours in subjects, preferably in woman presenting with a pelvic or ovarian mass e.g. pre-surgery, and to related methods and kits.
  • the invention is particularly important for assessing the risk of having or developing ovarian cancer in patients with an ovarian tumour or pelvic mass.
  • Ovarian cancer is the most common cause of death from gynaecological malignancies. It represents the fifth leading cause of death from all cancers for woman.
  • the non-specific clinical representation and the absence of effective screening methods are responsible for the 70% of patients who represent with an advanced stage of disease at the time of diagnosis. Since certainty about the nature of a pelvic or ovarian mass can only be acquired after histo- pathological examination, the traditional strategy for establishing the final diagnosis has been to perform an exploratory laparotomy.
  • CA125 has been evaluated as a discriminative tool to differentiate between benign and malignant ovarian cysts (cf. also the risk of malignancy index or RMI).
  • RMI malignancy index
  • CA125 is elevated in many benign gynaecological diseases that commonly affect premenopausal women such as endometriosis and in many medical conditions that affect postmenopausal women resulting in a reduction of specificity. These limitations have prompted the need to develop biomarkers with better sensitivity and specificity such as HE4.
  • the serum biomarker HE4 is consistently expressed in patients with ovarian cancer and has demonstrated an increased sensitivity and specificity over that of CA125 alone.
  • the predictive model ROMA (Risk of Ovarian Malignancy Algorithm) utilizes the combination of HE4 and CA125 values and provides a more sensitive tool to assess the risk of epithelial ovarian cancer in women with a pelvic or ovarian mass compared with either biomarker alone.
  • An alternative test is the OVA1 test which measures serum levels of CA125-II, transthyretin, apolipoprotein A1 , beta2-microglobulin and transferrin.
  • OVA1 test which measures serum levels of CA125-II, transthyretin, apolipoprotein A1 , beta2-microglobulin and transferrin.
  • none of the available biomarkers or tests adequately distinguishes benign from malignant ovarian cancers (Miller et al., Obstetrics & Gynecology 1 17, 1-9,
  • RMI Risk of Malignancy Index
  • Jacobs et al. 1990 (Br J Obstet. Gynaecol 1990;97:922-9), which is an algorithm that employs ultrasound (US) findings and architectural features of a pelvic mass, CA125 levels, and menopausal status.
  • US ultrasound
  • Several subsequent reports have validated the predicted levels of sensitivity and specificity (cf. Bailey J et al., 2006, Int J Gynecol Cancer 2006;16(Suppl):30-4; Manjunath AP et al., 2001 , Gynecol.
  • the RMI is a straightforward and widely used algorithm that produces a numeric score to stratify patients into high- and low-risk groups for EOC.
  • the RMI successfully categorizes patients into high- and low-risk groups, but it uses US imaging data that can have interpreter variability between users and centres.
  • Equally important, clinical evaluation of a pelvic mass often includes computed tomography (CT) imaging, magnetic resonance imaging (MRI), US, or a combination of imaging modalities resulting in a lack of standardization across imaging methods for risk of ovarian malignancy.
  • CT computed tomography
  • MRI magnetic resonance imaging
  • US or a combination of imaging modalities resulting in a lack of standardization across imaging methods for risk of ovarian malignancy.
  • the ROMA test was compared with the standard Risk of Malignancy Index (RMI), indicating that the ROMA test clearly outperformed the RMI test (Moore et al., 2010, Am J Obstet Gynecol 2010;203:228.e1-6). Yet, the ROMA test still has low specificity for distinguishing benign ovarian masses, cysts or tumours from early stage ovarian cancers or borderline cancers (Low Malignant Potential or LMPs).
  • RMI Risk of Malignancy Index
  • ovarian cancer In ovarian cancer, a favourable outcome of the disease is strongly correlated with accurate classification of the pelvic mass or ovarian cyst detected.
  • ovarian cancer patients treated by a specialized gynaecologic oncologist have fewer complications and better survival rates (Earle CC, et al., 2006, J Natl Cancer Institute; 98 (3): 172-80). To date however only 50% of patients with ovarian cancer receive this optimal debulking surgery.
  • the present invention addresses the above needs in the art by identifying biomarkers for determining the malignancy of ovarian cysts or pelvic masses and providing uses therefore.
  • Cystatin-C is also a well known filtration marker and a non-cancer related reduced kidney functioning can hamper the performance of the Cystatin-C marker in predicting or prognosis of ovarian cancer. For this reason, taking into account the eGfr value can be helpful to correct for the filtration function and hence to "normalise” or “correct” the Cystatin-C levels, thereby increasing its performance in the diagnosis of ovarian cancer.
  • the inventors have identified Cystatin-C or their fragments as new biomarkers advantageous for classifying ovarian tumours in a subject.
  • Cystatin-C or fragments thereof in combination with CA125 as a biomarker for classifying ovarian tumours in a subject.
  • the Cystatin-C level or quantity in the sample of the subject is normalized or corrected by means of the estimated Glomerular filtration rate (eGfr) in a sample of said subject.
  • the use of Cystatin-C or a fragment thereof in combination with HE4 as a biomarker for classifying ovarian tumours in a subject.
  • the Cystatin-C level or quantity in the sample of the subject is normalized or corrected by means of the estimated Glomerular filtration rate (eGfr) in a sample of said subject.
  • Cystatin-C or fragments thereof is provided in combination with both CA125 and HE4 for classifying ovarian tumours in a subject.
  • the Cystatin-C level or quantity in the sample of the subject is normalized or corrected by means of the estimated Glomerular filtration rate (eGfr) in a sample of said subject.
  • Cystatin-C or fragments thereof can be used for stratifying said subject for surgery.
  • the Cystatin-C level or quantity in the sample of the subject is normalized or corrected by means of the estimated Glomerular filtration rate (eGfr) in a sample of said subject.
  • the median AUC value (area under the ROC curve; "ROC” stands for receiver operating characteristic) in the analysis performed with Cystatin C alone was generally around 0.80, but can be increased to 0.95 when combine with other markers or parameters selected from HE4, CA125 and/or eGfr.
  • the AUC value is a combined measure of sensitivity and specificity and a higher AUC value (i.e., approaching 1 if the test has a sensitivity and specificity of 100%) in general indicates an improved performance of the test.
  • a method for classifying an ovarian tumour in a subject comprising measuring the quantity of Cystatin-C or fragments thereof in a sample from said subject.
  • measuring the levels of Cystatin-C or fragments thereof and/or other biomarker(s) in a sample from a subject may particularly denote that the examination phase of a method comprises measuring the quantity of Cystatin-C or fragments thereof thereof and/or other biomarker(s) in the sample from the subject.
  • methods of classification of an ovarian tumour generally comprise an examination phase in which data is collected from and/or about the subject.
  • the Cystatin-C level or quantity in the sample of the subject is normalized or corrected by means of the estimated Glomerular filtration rate (eGfr) in a sample of said subject.
  • the subject with an ovarian tumour can be a post-menopausal or a pre-menopausal woman.
  • the method as disclosed herein is for the classification of an ovarian tumour from a postmenopausal woman.
  • a method for classifying an ovarian tumour in a subject comprises: (i) measuring the quantity of Cystatin-C or fragments thereof in a sample from the subject; (ii) comparing the quantity of Cystatin-C or fragments thereof as measured in (i) with a reference value of the quantity of Cystatin-C or fragments thereof, said reference value representing a known classification of an ovarian tumour; (iii) finding a deviation or no deviation of the quantity of Cystatin-C or fragments thereof as measured in (i) from said reference value; (iv) classifying the ovarian tumour in said subject as being benign or malignant based on said finding of deviation or no deviation.
  • the Cystatin-C level or quantity in the sample of the subject is normalized or corrected by means of the estimated Glomerular filtration rate (eGfr) in a sample of said subject.
  • the method allows for a reliable classification and hence diagnosis of an ovarian tumour in a subject, in particular in a woman presenting with a pelvic mass pre-surgery.
  • an altered quantity (i.e., a deviation) of Cystatin-C or fragments thereof in a sample from a subject compared to a reference value representing the classification of a benign ovarian cyst seems to indicate that the subject has or is at risk of having a malignant ovarian cancer.
  • a comparable quantity (i.e., no deviation) of Cystatin-C or fragments thereof in a sample from a subject compared to a reference value representing the classification of a benign ovarian cyst indicates that the subject has or has a chance of having a benign ovarian cyst.
  • the Cystatin-C level or quantity in the sample of the subject is normalized or corrected by means of the estimated Glomerular filtration rate (eGfr) in a sample of said subject.
  • an altered quantity (i.e., a deviation) of Cystatin-C or fragments thereof in a sample from a subject compared to a reference value representing the classification of a malignant ovarian cancer indicates that the subject has or has a chance of having a benign ovarian cyst.
  • a comparable quantity (i.e., no deviation) of Cystatin-C or fragments thereof in a sample from a subject compared to a reference value representing the classification of a malignant ovarian cancer indicates that the subject has or is at risk of having a malignant ovarian cyst.
  • the Cystatin-C level or quantity in the sample of the subject is normalized or corrected by means of the estimated Glomerular filtration rate (eGfr) in a sample of said subject.
  • a method for the classification of an ovarian tumour in a subject, wherein an altered quantity of Cystatin-C or fragments thereof in a sample from a subject compared to a reference value representing the classification of a benign ovarian cyst indicates that the subject has or is at risk of having a malignant ovarian cancer. Furthermore, a method is disclosed for the classification of an ovarian tumour in a subject, wherein a comparable quantity of Cystatin-C or fragments thereof in a sample from a subject compared to a reference value representing the classification of a benign ovarian cyst indicates that the subject has or has a chance of having a benign ovarian cyst.
  • the Cystatin-C level or quantity in the sample of the subject is normalized or corrected by means of the estimated Glomerular filtration rate (eGfr) in a sample of said subject.
  • a method for the classification of an ovarian tumour in a subject wherein an altered quantity of Cystatin-C or fragments thereof in a sample from a subject compared to a reference value representing the classification of a malignant ovarian cancer indicates that the subject has or has a chance of having a benign ovarian cyst.
  • a method for the classification of an ovarian tumour in a subject wherein a comparable quantity of Cystatin- C or fragments thereof in a sample from a subject compared to a reference value representing the classification of a malignant ovarian cancer indicates that the subject has or is at risk of having a malignant ovarian cancer.
  • the Cystatin-C level or quantity in the sample of the subject is normalized or corrected by means of the estimated Glomerular filtration rate (eGfr) in a sample of said subject.
  • the ovarian tumours classified with the methods as described herein can be epithelial carcinoma, sex cord carcinoma, germ cell carcinoma, cystadenoma, fibroma, thecoma, cystadenofibroma, mature teratoma, endometriosis, follicular cyst, abces, struma ovarii, Leydig cell tumour, parasalpingeal cyst, hydrosalpinx, corpus luteum cyst, heamorragic cyst, tissue with calcifications NOS, necrotic tumour NOS, metastatic cancers infiltrated from other tissues, or combinations thereof.
  • the ovarian cysts classified as benign with the methods as described herein can be cystadenoma, fibroma, thecoma, cystadenofibroma, mature teratoma, endometriosis, follicular cyst, abces, struma ovarii, Leydig cell tumour, parasalpingeal cyst, hydrosalpinx, corpus luteum cyst, heamorragic cyst, tissue with calcifications NOS, necrotic tumour NOS or combinations thereof.
  • the ovarian cancers classified as malignant with the methods as described herein can be epithelial carcinoma, sex cord carcinoma, germ cell carcinoma, metastatic cancers infiltrated from other tissues, or combinations thereof.
  • tumour subclass may be selected from the group comprising serous epithelial carcinoma, endometroid epithelial carcinoma, mucinous epithelial carcinoma, clear cell epithelial carcinoma, Brenner epithelial carcinoma, carcinosarcoma, undifferentiated epithelial carcinoma, granulosa sex cord carcinoma, sertoli-leydig sex cord carcinoma, gyandroblastoma, dysgerminoma, yolk sac carcinoma, embryonal carcinoma, choriocarcinoma, immature teratoma, serous cystadenoma, endometrioid cystadenoma, mucinous cystadenoma, clear cell cystadenoma, Brenner cystadenoma, fibroma, thecoma, fribrothecoma, of serous cystadenofibroma
  • a method for classifying an ovarian tumour in a subject comprising: (i) measuring the quantity of Cystatin-C or fragments thereof in a sample from the subject; (ii) comparing the quantity of Cystatin-C or fragments thereof as measured in (i) with a reference value of the quantity of Cystatin-C or fragments thereof, said reference value representing a known classification of an ovarian tumour; (iii) finding a deviation or no deviation of the quantity of Cystatin-C or fragments thereof as measured in (i) from said reference value; (iv) classifying the ovarian tumour in said subject as belonging to a subclass of ovarian tumours based on said finding of deviation or no deviation.
  • the Cystatin-C level or quantity in the sample of the subject is normalized or corrected by means of the estimated Glomerular filtration rate (eGfr) in a sample of said subject.
  • the methods may also be used to detect various stages or progression or severity such as grading of the ovarian cancer.
  • the method for classifying an ovarian tumour in a subject may be performed for a subject at two or more successive time points and the respective outcomes at said successive time points may be compared, whereby the presence or absence of a change between the classification of an ovarian tumour in a subject at said successive time points is determined.
  • the method thus allows monitoring a change in the classification of an ovarian tumour in a subject over time.
  • the methods for classifying an ovarian tumour in a subject may further comprise the step of stratifying said subject for surgery based on the results of the tumour classification step.
  • a subject with an ovarian tumour may be stratified for surgery for instance by a gynaecologist, where the tumour is classified as benign.
  • a subject with an ovarian tumour may be stratified for surgery for instance by an oncologic or gynaecologic surgeon, where the tumour is classified as being malignant. Therefore, the methods may be used to help the medical practitioner to decide upon the course of therapy and in particular upon the surgery strategy.
  • the type of operative procedure and the following chemotherapy will also depend on the stratification of said subject based on the tumour classification and patient stratification.
  • a method to stratify a subject for the risk of having or developing an ovarian tumour or cancer comprising: (i) measuring the quantity of Cystatin-C or fragments thereof in a sample from the subject; (ii) comparing the quantity of Cystatin-C or fragments thereof as measured in (i) with a reference value of the quantity of Cystatin-C or fragments thereof, said reference value representing a known classification of an ovarian tumour or cancer; (iii) finding a deviation or no deviation of the quantity of Cystatin-C or fragments thereof as measured in (i) from said reference value; (iv) classifying the ovarian tumour or cancer in said subject as being benign or malignant based on said finding of deviation or no deviation; (v) optionally stratifying said subject for surgery based on the results of the classification as obtained in (iv).
  • the Cystatin-C level or quantity in the sample of the subject is normalized or corrected by means of the estimated Glomerular filtration rate (eGfr) in a sample of said subject.
  • the present methods may be preferably performed in a subject once the subject is diagnosed with an ovarian tumour or a pelvic, adnexal, or ovarian mass, or cyst, more preferably before surgery of the subject.
  • the methods for the classification of an ovarian tumour or cyst in a subject or for the risk stratification of a subject with an ovarian tumour or cyst may be performed for a subject immediately before or close to the surgery, but can also be performed several months before surgery, preferably less than 50 days before surgery; preferably, the classification and/or stratification methods as described herein may be performed for a subject less than 5 days before surgery.
  • the measurement of Cystatin-C or fragments thereof may also be combined with the assessment of CA125, and/or HE4, optionally with one or more further biomarkers or clinical parameters relevant for the ovarian tumours, such as the eGfr, based on creatinine levels in blood or urine.
  • the examination phase of the methods further (i.e. in addition to measuring Cystatin-C or fragments thereof) comprises measuring the quantity of CA125, HE4 and/or analyzing the eGfr parameter, and/or one or more such other markers or parameters in the sample from the subject.
  • any known or yet unknown suitable marker could be used.
  • biomarkers "other than Cystatin-C” or “other biomarkers” generally encompasses such other biomarkers which are useful for the classification of ovarian cysts as disclosed herein.
  • biomarkers useful for the classification of an ovarian tumour in a subject include mesothelin, CA72-4, osteopontin and inhibin, and fragments or precursors of any one thereof.
  • the methods for classifying and stratifying as defined according to the present invention can also include or be combined with clinical parameters for evaluating the malignancy or risk thereto of pelvic masses.
  • ultrasound, (US), magnetic resonance imaging (MRI) or computed tomography (CT) can be used to assess the morphology of the cyst(s), and/or the presence of multi-locular cysts, evidence of solid areas, evidence of metastases, presence of ascites, and/or bilateral lesions.
  • the menopausal status of the subject can be taken into account.
  • the Risk of Malignancy Index (RMI) for example combines these clinical parameters with the serum level of CA125 in the subject resulting in a risk score.
  • the methods as described herein may further (i.e. in addition to measuring Cystatin-C or fragments thereof) comprise measuring the quantity of CA125 as a biomarker in the sample from said subject.
  • the methods as described herein may further (i.e. in addition to measuring Cystatin-C or fragments thereof) comprise measuring the quantity of HE4 as a biomarker in the sample from said subject.
  • the methods as described herein may also further (i.e.
  • Cystatin-C or fragments thereof in addition to measuring Cystatin-C or fragments thereof) comprise measuring the quantity of both CA125 and HE4 as biomarkers in the sample from said subject.
  • the Cystatin-C level or quantity in the sample of the subject is normalized or corrected by means of the estimated Glomerular filtration rate (eGfr) in a sample of said subject.
  • the method for classifying an ovarian tumour in a subject may then comprise: (i) measuring the quantity of Cystatin-C or fragments thereof and the quantity of CA125 and HE4 in a sample from the subject; (ii) using the measurements of (i) to establish a subject profile of the quantity of Cystatin-C or fragments thereof and the quantity of CA125 and HE4; (iii) comparing said subject profile of (ii) to a reference profile of the quantity of Cystatin-C or fragments thereof and the quantity of CA125 and HE4, said reference profile representing a known classification of an ovarian tumour; (iv) finding a deviation or no deviation of the subject profile of (ii) from the reference profile; (v) classifying the ovarian tumour in said subject as being benign or malignant based on said finding of deviation or no deviation.
  • the Cystatin-C level or quantity in the sample of the subject is normalized or corrected by means of the estimated Glomerular filtration rate (eGfr) in a sample of said
  • an altered quantity (i.e., a deviation) of Cystatin-C or fragments thereof and an altered quantity (i.e., a deviation) of CA125 and/or HE4 in a sample from a subject compared to a reference value representing the classification of a benign ovarian cyst indicates that the subject has or is at risk of having a malignant ovarian cancer.
  • an altered quantity (i.e., a deviation) of Cystatin-C or fragments thereof and an altered quantity (i.e., a deviation) of CA125 and/or HE4 in a sample from a subject compared to a reference value representing the classification of a malignant ovarian cancer indicates that the subject has or has a chance of having a benign ovarian cyst.
  • the Cystatin-C level or quantity in the sample of the subject is normalized or corrected by means of the estimated Glomerular filtration rate (eGfr) in a sample of said subject.
  • the method for stratifying a subject with a pelvic mass for the risk of having or developing ovarian cancer comprising: (i) measuring the quantity of Cystatin-C or fragments thereof and the quantity of CA125 and HE4 in a sample from the subject; (ii) using the measurements of (i) to establish a subject profile of the quantity of Cystatin-C or fragments thereof and the quantity of CA125 and HE4; (iii) comparing said subject profile of (ii) to a reference profile of the quantity of Cystatin-C or fragments thereof and the quantity of CA125 and HE4, said reference profile representing a known classification of an ovarian tumour; (iv) finding a deviation or no deviation of the subject profile of (ii) from the reference profile; (v) classifying the ovarian tumour in said subject as being benign or malignant based on said finding of deviation or no deviation; (vi) stratifying said subject for surgery based on the results of the classification as obtained in (v).
  • Cystatin-C level or quantity in the sample of the subject is normalized or corrected by means of the estimated Glomerular filtration rate (eGfr) in a sample of said subject.
  • Cystatin-C or fragments thereof in combination with CA125 and/or HE4 can also be used for monitoring subjects with ovarian cancer post-surgery.
  • the Cystatin-C level or quantity in the sample of the subject is normalized or corrected by means of the estimated Glomerular filtration rate (eGfr) in a sample of said subject.
  • Cystatin-C or fragments thereof in combination with CA125 and/or HE4 can further be used for screening of subjects belonging to a high risk group for ovarian cancer.
  • the Cystatin-C level or quantity in the sample of the subject is normalized or corrected by means of the estimated Glomerular filtration rate (eGfr) in a sample of said subject.
  • Subjects belonging to a high risk group for ovarian cancer can be for instance patients who have previously undergone surgery of an ovarian tumour, post-menopausal woman receiving hormone or hormone replacement treatment, or subjects with genetic predisposition for ovarian cancer.
  • Genetic predisposition is an importance risk factor for ovarian cancer.
  • Women with two or more first-degree relatives with ovarian cancer have as much as 50% risk of becoming affected (Freddo, West J Med, 1992, 156(6): 652).
  • certain mutations in the BRCA-family of genes (BRCA-1 , BRCA-2, BRCA-3, BRCA-X or other genes, such as RAD51 D) have been reported to occur in patients that have a high risk of developing Breast, ovarian, or prostate cancer.
  • Subjects having such mutations therefore belong to a high risk group for developing ovarian cancer.
  • Other factors influencing the risk for ovarian cancer include inter alia age, post-menopausal status, diet, obesity, reproductive history, gynaecological surgery such as tubal ligation and hysterectomy, hormonal (replacement) therapy, smoking and alcohol use.
  • Cystatin-C or fragments thereof in combination with CA125 and/or HE4 for screening of subjects belonging to a high risk group can be useful for monitoring the recurrence or occurrence of an ovarian tumour in said subjects belonging to a high risk group.
  • the Cystatin-C level or quantity in the sample of the subject is normalized or corrected by means of the estimated Glomerular filtration rate (eGfr) in a sample of said subject.
  • a method for monitoring a subject with ovarian cancer post-surgery comprising: (i) measuring the quantity of Cystatin-C or fragments thereof and the quantity of CA125 and/or HE4 value in a sample from the subject; (ii) using the measurements of (i) to establish a subject profile of the quantity of Cystatin-C or fragments thereof and the quantity of CA125 and/or HE4; (iii) comparing said subject profile of (ii) to a reference profile of the quantity of Cystatin-C or fragments thereof and the quantity of CA125 and/or HE4 said reference profile representing a known quantity of Cystatin-C or fragments thereof and a known quantity of CA125 and/or HE4 in a sample from the subject pre-surgery; (iv) finding a deviation or no deviation of the subject profile of (ii) from the reference profile; (v) attributing said finding of deviation or no deviation to a particular prognosis for ovarian cancer in said subject.
  • a method for screening a subject belonging to a high risk group for ovarian cancer comprising: (i) measuring the quantity of Cystatin-C or fragments thereof and the quantity of CA125 and/or HE4in a sample from the subject; (ii) using the measurements of (i) to establish a subject profile of the quantity of Cystatin-C or fragments thereof and the quantity of CA125 and/or HE4; (iii) comparing said subject profile of (ii) to a reference profile of the quantity of Cystatin-C or fragments thereof and the quantity of CA125 and/or HE4, said reference profile representing a known quantity of Cystatin-C or fragments thereof and a known quantity of CA125 and/or HE4 in a sample from a healthy subject; (iv) finding a deviation or no deviation of the subject profile of (ii) from the reference profile; (v) attributing said finding of deviation or no deviation to a particular risk for having or developing ovarian cancer in said subject.
  • a method for screening a subject belonging to a high risk group for ovarian cancer may be performed for said subject belonging to a high risk group for ovarian cancer at two or more successive time points and the respective outcomes at said successive time points may be compared, whereby the presence or absence of a change between said successive time points is determined.
  • the method thus allows monitoring the risk of ovarian cancer in said subject over time.
  • a method for screening a subject belonging to a high risk group for ovarian cancer comprising: (i) measuring the quantity of Cystatin-C or fragments thereof and the quantity of CA125 and/or HE4in a sample from the subject from two or more successive time points; (ii) comparing the quantity of Cystatin-C or fragments thereof and the quantity of CA125 and/or HE4between the samples as measured in (i); (iii) finding the rate of change of the quantity of Cystatin-C or fragments thereof between the samples as compared in (ii); and (iv) attributing said finding of rate of change between the two or more successive time points to a change in the risk for ovarian cancer in the subject.
  • the Cystatin-C level or quantity in the sample of the subject is normalized or corrected by means of the estimated Glomerular filtration rate (eGfr) in a sample of said subject.
  • an altered quantity (i.e., a deviation) of Cystatin-C or fragments thereof, optionally compensated by the measured eGfr value, and an altered quantity (i.e., a deviation) of CA125, HE4 between the samples from said subject at two successive time points indicates that the subject has a changed risk for having or developing ovarian cancer.
  • the methods as described herein may further (i.e. in addition to measuring Cystatin-C or fragments thereof) comprise measuring the quantity of one or more other biomarkers in the sample from said subject, wherein said other biomarker is chosen from the group consisting of mesothelin, CA72-4, osteopontin and inhibin, and fragments or precursors of any one thereof.
  • the method for classifying an ovarian tumour in a subject may then comprise: (i) measuring the quantity of Cystatin-C or fragments thereof and the quantity of CA125, HE4 and/or one or more other biomarkers in a sample from the subject; (ii) using the measurements of (i) to establish a subject profile of the quantity of Cystatin-C or fragments thereof and the quantity of CA125, HE4 and/or one or more other biomarkers; (iii) comparing said subject profile of (ii) to a reference profile of the quantity of Cystatin-C or fragments thereof and the quantity of CA125, HE4 and/or one or more other biomarkers, said reference profile representing a known classification of an ovarian tumour; (iv) finding a deviation or no deviation of the subject profile of (ii) from the reference profile; (v) classifying the ovarian tumour in said subject as being benign or malignant based on said finding of deviation or no deviation.
  • the Cystatin-C value can be compensated
  • the method for stratifying a subject with an ovarian tumour may comprise: (i) measuring the quantity of Cystatin-C or fragments thereof and the quantity of CA125, HE4 and/or one or more other biomarkers in a sample from the subject; (ii) using the measurements of (i) to establish a subject profile of the quantity of Cystatin-C or fragments thereof and the quantity of CA125, HE4 and/or one or more other biomarkers; (iii) comparing said subject profile of (ii) to a reference profile of the quantity of Cystatin-C or fragments thereof and the quantity of CA125, HE4 and/or one or more other biomarkers, said reference profile representing a known classification of an ovarian tumour; (iv) finding a deviation or no deviation of the subject profile of (ii) from the reference profile; (v) classifying the ovarian tumour in said subject as being benign or malignant based on said finding of deviation or no deviation; (vi) stratifying said subject for surgery based on the results of the results of
  • Any one classification and/or stratification method as taught herein may preferably allow for sensitivity and/or specificity (preferably, sensitivity and specificity) of at least 50%, at least 60%, at least 70% or at least 80%, e.g., ⁇ 85% or > 90% or >95%, e.g., between about 80% and 100% or between about 85% and 95%.
  • sensitivity and/or specificity preferably, sensitivity and specificity
  • the respective quantities, measurements or scores for the biomarker(s) and (clinical) parameter(s) in the present test panels may be evaluated separately or individually, i.e., each compared with its corresponding reference value. More advantageously, the quantities, measurements or scores for the biomarker(s) and parameter(s) may be used to establish a biomarker or biomarker-and-parameter profile or value, which can be suitably compared with a corresponding reference profile or value.
  • the measured level(s) of biomarker(s) are not compared to a reference value, but are used in an equation, wherein each biomarker level is given a certain weight.
  • Said scale indicates the ranges of the numerical values pointing towards 1 ) benign, or 2) malignant ovarian tumours, preferably pointing towards 1 ) benign and 2) early malignant, or borderline (LMP) tumours.
  • LMP borderline
  • the quantities, measurements or scores for the biomarker(s) and parameter(s) may thus each be modulated by an appropriate weighing factor and added up to yield a single value, which can then be suitably compared with a corresponding reference value obtained accordingly.
  • the invention thus provides for a method for the classification of an ovarian tumour in a subject, comprising: (i) measuring the quantity of Cystatin-C or fragments thereof, optionally compensated by the measured eGfr value, and measuring the quantity of HE4 and/or CA125 in a sample from the subject; (ii) entering the quantities measured in (i) into an equation; and (iii), analysing whether the numerical value obtained in (ii) falls within the range of 1 ) benign or 2) malignant ovarian tumour, preferably within the range of 1 ) benign or 2) early malignant, or borderline (LMP) tumours.
  • the ranges of numerical values produced by the equation that reflect benign or malignant (preferably early stage malignant, or borderline) ovarian cancer can be established by measuring the quantity of Cystatin-C or fragments thereof, optionally compensated by the measured eGfr value, and measuring the quantity of HE4 and/or CA125 in a sample from a subject for which the ovarian cyst, tumour or cancer has been previously diagnosed or classified.
  • the invention thus also provides a risk stratification tool, based on the concentrations of Cystatin-C or fragments thereof, optionally compensated by the measured eGfr value, and the concentration of HE4 and/or CA125, optionally combined with clinical parameters, risk factors, or other biomarkers.
  • the risk stratification tool is an equation that yields a certain value, which can be projected on a scale, indicative for the risk of malignancy of the ovarian mass in a subject. Said scale is established using the same equation on samples of subjects which have already been diagnosed with a specific type of ovarian cyst, tumour or cancer.
  • Ultrasound US
  • Magnetic Resonance Imaging MRI
  • Computed Tomography CT
  • the concentration of one or more other biomarkers in the sample from said subject is chosen from the group consisting of mesothelin, CA72-4, osteopontin and inhibin, and fragments or precursors of any one thereof,
  • risk factors such as genetic (e.g. gene mutations in the BRCA genes) and/ or familial predisposition (e.g. direct family members with cancer), age, diet, obesity, reproductive history, menopausal status, gynaecological surgery such as tubal ligation and hysterectomy, hormonal (replacement) therapy, smoking and alcohol use.
  • said risk stratification tool uses the concentration of Cystatin-C or fragments thereof, optionally compensated by the measured eGfr value, and the concentration of CA125 and HE4 in a sample of the subject, each with their own weight factors.
  • said risk stratification tool uses the concentration of Cystatin-C or fragments thereof, optionally compensated by the measured eGfr value, and the concentration of CA125 and HE4 in a sample of the subject combined with the clinical and morphological analysis of the pelvic mass, through e.g. Ultrasound (US), Magnetic Resonance Imaging (MRI), Computed Tomography (CT), or any combination of said imaging methods, each with their own weight factors.
  • US Ultrasound
  • MRI Magnetic Resonance Imaging
  • CT Computed Tomography
  • weighing factors may depend on the methodology used to quantify biomarkers and measure or score parameters, and for each particular experimental setting may be determined and comprised in a model suitable for diagnosis, prediction and/or prognosis of the diseases and conditions as taught herein.
  • Various methods can be used for the purpose of establishing such models, e.g., support vector machine, Bayes classifiers, logistic regression, etc. (Cruz et al. Applications of Machine Learning in Cancer Prediction and Prognosis. Cancer Informatics 2007; 2; 59-77).
  • Reference values as employed herein may be established according to known procedures previously employed for other test panels comprising biomarkers and/or clinical parameters. Reference values may be established either within (i.e., constituting a step of) or external to (i.e., not constituting a step of) the methods and uses as taught herein. Accordingly, any one of the methods or uses taught herein may comprise a step of establishing a requisite reference value.
  • the present methods may employ reference values for the quantity of Cystatin-C or fragments thereof, which may be established according to known procedures previously employed for other biomarkers. Such reference values may be established either within (i.e., constituting a step of) or external to (i.e., not constituting a step of) the methods of the present invention as defined herein.
  • any one of the methods taught herein may comprise a step of establishing a reference value for the quantity of Cystatin-C or fragments thereof, said reference value representing either (a) a classification of the ovarian cyst as being benign or (b) a classification of the ovarian cyst as being malignant.
  • the Cystatin-C value can be compensated or corrected by the measured eGfr value.
  • a further aspect provides a method for establishing a reference value for the quantity of Cystatin-C or fragments thereof, said reference value representing:
  • the present methods may otherwise employ reference profiles for the quantity of Cystatin-C or fragments thereof and the quantity of one or more other biomarkers, which may be established according to known procedures previously employed for other biomarkers. Such reference profiles may be established either within (i.e., constituting a step of) or external to (i.e., not constituting a step of) the present methods. Accordingly, the methods taught herein may comprise a step of establishing a reference profile for the quantity of Cystatin-C or fragments thereof and the quantity of said one or more other biomarkers, said reference profile representing either (a) a classification of an ovarian cyst as being benign, or (b) a classification of an ovarian cancer as being malignant.
  • the Cystatin-C value can be compensated or corrected by the measured eGfr value.
  • a further aspect provides a method for establishing a reference profile for the quantity of Cystatin-C or fragments thereof and the quantity of CA125, HE4 and/or one or more other biomarkers useful for the classification of an ovarian tumour in a subject as taught herein, said reference profile representing:
  • a method for establishing a Cystatin-C or fragments thereof base-line or reference value in a subject comprising: (i) measuring the quantity of Cystatin-C or fragments thereof in the sample from the subject at different time points wherein the subject is not suffering from the diseases or conditions as taught herein, and (ii) calculating the range or mean value of the subject, which is the Cystatin-C or fragments thereof base-line or reference value for said subject.
  • the Cystatin-C value can be compensated or corrected by the measured eGfr value.
  • the subject as intended in any one of the present methods may be human, preferably, pre- or post-menopausal, more preferably, post menopausal.
  • said sample is blood, serum or plasma.
  • said classifying of tumours includes distinguishing benign tumours from malignant cancers, more preferably, said classifying includes distinguishing benign tumours from early stage ovarian cancers and cancers with low malignant potential (LMPs).
  • LMPs low malignant potential
  • said ovarian tumour is an epithelial carcinoma, sex cord carcinoma, germ cell carcinoma, metastatic carcinoma infiltrated in the pelvis or in the ovaries, cystadenoma, fibroma, thecoma, cystadenofibroma, mature teratoma, endometriosis, follicular cyst, abces, struma ovarii, Leydig cell tumour, parasalpingeal cyst, hydrosalpinx, corpus luteum cyst, heamorragic cyst, tissue with calcifications NOS, necrotic tumour NOS or combinations thereof.
  • kits for performing the methods according to the present invention comprising: (i) means for measuring the quantity of Cystatin-C or a fragment thereof and the quantity of CA125, HE4 and/or one or more other biomarkers in a sample from the subject, and preferably further comprising (ii) a reference value of the quantity of Cystatin-C or fragments thereof and the quantity of CA125, HE4 and/or one or more other biomarkers or means for establishing said reference value, wherein said reference value represents a known classification of an ovarian tumour.
  • said quantity of Cystatin-C or fragments thereof and/or the quantity of CA125, HE4 and/or one or more other biomarkers may be measured by any suitable technique such as may be known in the art.
  • the quantity of Cystatin-C or fragments thereof and/or the quantity of CA125, HE4 and/or one or more other biomarkers may be measured using, respectively, a binding agent capable of specifically binding to Cystatin-C or fragments thereof and/or to fragments thereof, and a binding agent capable of specifically binding to CA125, HE4 and/or one or more other biomarkers.
  • the binding agent may be an antibody, aptamer, photoaptamer, protein, peptide, peptidomimetic or a small molecule.
  • the quantity of Cystatin-C or fragments thereof and/or the quantity of CA125, HE4 and/or one or more other biomarkers may be measured using an immunoassay technology or a mass spectrometry analysis method or a chromatography method, or a combination of said methods.
  • said quantity of Cystatin-C or fragments thereof and/or the quantity of CA125, HE4 and/or one or more other biomarkers is measured using an immunoassay technology, or using a mass spectrometry analysis method or using a chromatography method, or using a combination of said methods, or using RNA analysis tools such as northern blotting, or (quantitative) RT-PCR.
  • kits for classifying an ovarian tumour in a subject comprising (i) means for measuring the quantity of Cystatin-C or fragments thereof in a sample from the subject, and optionally and preferably (ii) a reference value of the quantity of Cystatin-C or fragments thereof or means for establishing said reference value, wherein said reference value represents a known classification of an ovarian tumour.
  • the kit thus allows one to: measure the quantity of Cystatin-C or fragments thereof in the sample from the subject by means (i); compare the quantity of Cystatin-C or fragments thereof measured by means (i) with the reference value of (ii) or established by means (ii); find a deviation or no deviation of the quantity of Cystatin-C or fragments thereof measured by means (i) from the reference value of (ii); and consequently attribute said finding of deviation or no deviation to a particular classification of the ovarian tumour in the subject.
  • the Cystatin-C value can be compensated or corrected by the measured eGfr value.
  • a further embodiment provides a kit for classifying an ovarian tumour in a subject as taught herein, the kit comprising (i) means for measuring the quantity of Cystatin-C or fragments thereof in a sample from the subject and (ii) means for measuring the quantity of CA125, HE4 and/or one or more other biomarkers in the sample from the subject, and optionally and preferably (iii) means for establishing a subject profile of the quantity of Cystatin-C or fragments thereof and the quantity of CA125, HE4 and/or one or more other biomarkers, and optionally and preferably (iv) a reference profile of the quantity of Cystatin-C or fragments thereof and the quantity of CA125, HE4 and/or one or more other biomarkers, or means for establishing said reference profile, said reference profile representing a known classification of an ovarian tumour.
  • Such kit thus allows one to: measure the quantity of Cystatin-C or fragments thereof and the quantity of CA125, HE4 and/or one or more other biomarkers in the sample from the subject by respectively means (i) and (ii); establish (e.g., using means included in the kit or using suitable external means) a subject profile of the quantity of Cystatin-C or fragments thereof and the quantity of CA125, HE4 and/or one or more other biomarkers based on said measurements; compare the subject profile with the reference profile of (iv) or established by means (iv); find a deviation or no deviation of said subject profile from said reference profile; and consequently attribute said finding of deviation or no deviation to a particular classification of the ovarian tumour in the subject.
  • the Cystatin-C value can be compensated or corrected by the measured eGfr value.
  • the means for measuring the quantity of Cystatin-C or fragments thereof and/or the quantity of CA125, HE4 and/or one or more other biomarkers in the present kits may comprise, respectively, one or more binding agents capable of specifically binding to Cystatin-C or fragments thereof, and one or more binding agents capable of specifically binding to said CA125, HE4 and/or one or more other biomarkers.
  • any one of said one or more binding agents may be an antibody, aptamer, photoaptamer, protein, peptide, peptidomimetic or a small molecule.
  • any one of said CA125, HE4 and/or one or more other biomarkers may be advantageously immobilised on a solid phase or support.
  • the means for measuring the quantity of Cystatin-C or fragments thereof and/or the quantity of CA125, HE4 and/or one or more other biomarkers in the present kits may employ an immunoassay technology or mass spectrometry analysis technology or chromatography technology, or a combination of said technologies.
  • kits for the classification of an ovarian tumour in a subject comprising: (a) one or more binding agents capable of specifically binding to Cystatin-C or fragments thereof; (b) preferably, a known quantity or concentration of Cystatin-C or fragments thereof (e.g., for use as controls, standards and/or calibrators); (c) preferably, a reference value of the quantity of Cystatin-C or fragments thereof, or means for establishing said reference value.
  • binding agents capable of specifically binding to Cystatin-C or fragments thereof
  • a known quantity or concentration of Cystatin-C or fragments thereof e.g., for use as controls, standards and/or calibrators
  • c preferably, a reference value of the quantity of Cystatin-C or fragments thereof, or means for establishing said reference value.
  • Said components under (a) and/or (c) may be suitably labelled as taught elsewhere in this specification.
  • kits for the classification of an ovarian tumour in a subject comprising: (a) one or more binding agents capable of specifically binding to Cystatin-C or fragments thereof; (b) one or more binding agents capable of specifically binding to CA125, HE4 and/or one or more other biomarkers; (c) preferably, a known quantity or concentration of Cystatin-C or fragments thereof and a known quantity or concentration of said CA125, HE4 and/or one or more other biomarkers (e.g., for use as controls, standards and/or calibrators); (d) preferably, a reference profile of the quantity of Cystatin-C or fragments thereof and the quantity of said CA125, HE4 and/or one or more other biomarkers, or means for establishing said reference profiles.
  • Said components under (a), (b) and/or (c) may be suitably labelled as taught elsewhere in this specification.
  • kit as described herein for classifying an ovarian tumour in a subject as being benign or malignant as taught herein. Also disclosed is the use of the kit as described herein for stratifying a subject with an ovarian tumour for the risk of having or developing ovarian cancer as taught herein.
  • reagents and tools useful for measuring Cystatin-C or fragments thereof and optionally CA125, HE4 and/or one or more other biomarkers concerned herein are also disclosed.
  • a protein, polypeptide or peptide array or microarray comprising (a) Cystatin-C or fragments thereof, preferably a known quantity or concentration of said Cystatin- C or fragments thereof; and (b) optionally and preferably, CA125, HE4 and/or one or more other biomarkers, preferably a known quantity or concentration of said CA125, HE4 and/or one or more other biomarkers, useful in detecting antibodies to said biomarker(s) in a sample of the subject.
  • eGfr value to compensate for aberrant or abnormal kidney functioning, which can disturb the performance of the biomarkers Cystatin-C, HE4, and/or CA125, as used herein for diagnosis, prognosis, or classification of ovarian tumours or pelvic masses as defined herein.
  • binding agent array or microarray comprising: (a) one or more binding agents capable of specifically binding to Cystatin-C or fragments thereof, preferably a known quantity or concentration of said binding agents; and (b) optionally and preferably, one or more binding agents capable of specifically binding to CA125, HE4 and/or one or more other biomarkers, preferably a known quantity or concentration of said binding agents.
  • kits and devices in the methods for classifying tumours, risk stratifying, screening, and/or monitoring subjects as defined herein is also envisaged by the present invention.
  • Cystatin-C or fragments thereof may be a valuable target for therapeutic and/or prophylactic interventions in subjects with an ovarian tumour as described herein, in particular in subjects presenting with a pelvic mass pre-surgery or post- surgery, as specific chemotherapy. This is especially interesting since Cystatin-C has been implicated in angiogenesis inhibition.
  • agent is an antibody or a fragment or derivative thereof; a polypeptide; a peptide; a peptidomimetic; an aptamer; a photoaptamer; or a chemical substance, preferably an organic molecule, more preferably a small organic molecule.
  • (1 1 ) The agent isolated by the assay as set forth in (10) above.
  • a pharmaceutical composition or formulation comprising a prophylactically and/or therapeutically effective amount of one or more agents as set forth in any one of (1 ) to (8) or (10) above, or a pharmaceutically acceptable N-oxide form, addition salt, prodrug or solvate thereof, and further comprising one or more of pharmaceutically acceptable carriers.
  • Said ovarian tumours as set forth in any one of (1 ) to (13) above may be particularly chosen from epithelial carcinoma, sex cord carcinoma, germ cell carcinoma, infiltrated metastatic carcinoma, cystadenoma, fibroma, thecoma, cystadenofibroma, mature teratoma, endometriosis, follicular cyst, abces, struma ovarii, Leydig cell tumour, parasalpingeal cyst, hydrosalpinx, corpus luteum cyst, heamorragic cyst, tissue with calcifications NOS, necrotic tumour NOS or combinations thereof.
  • a screening method for selecting an agent capable of specifically binding to Cystatin-C or fragments thereof (e.g., gene or protein) comprising: (a) providing one or more, preferably a plurality of, test binding agents; (b) selecting from the test binding agents of (a) those which bind specifically to Cystatin-C or Creatinine; and (c) counter-selecting (i.e., removing) from the test binding agents selected in (b) those which bind to any one or more other, unintended or undesired, targets.
  • a screening assay for selecting an agent capable of specifically binding to Cystatin-C or fragments thereof (e.g., gene or protein) comprising: (a) providing one or more, preferably a plurality of, test binding agents; (b) selecting from the test binding agents of (a) those which bind specifically to Cystatin-C or Creatinine; and (c) counter-selecting (i.e., removing) from the test binding agents selected in (b) those
  • Binding between test binding agents and Cystatin-C may be advantageously tested by contacting (i.e., combining, exposing or incubating) said Cystatin-C with the test binding agents under conditions generally conducive for such binding.
  • binding between test binding agents and the Cystatin-C may be suitably tested in vitro; or may be tested in host cells or host organisms comprising the Cystatin-C and exposed to or configured to express the test binding agents.
  • the Cystatin-C binding or Cystatin-C modulating agents may be capable of binding to Cystatin-C or modulating the activity and/or level of the Cystatin-C in vitro, in a cell, in an organ and/or in an organism.
  • modulation of the activity and/or level of the Cystatin-C by test modulating agents may be advantageously tested by contacting (i.e., combining, exposing or incubating) said Cystatin-C (e.g., gene or protein) with the test modulating agents under conditions generally conducive for such modulation.
  • said Cystatin-C e.g., gene or protein
  • said conditions may be generally conducive for such binding.
  • modulation of the activity and/or level of the Cystatin-C by test modulating agents may be suitably tested in vitro; or may be tested in host cells or host organisms comprising the Cystatin-C and exposed to or configured to express the test modulating agents.
  • Cystatin-C or fragments thereof for use as a medicament, preferably for use in the treatment of ovarian tumours as described herein;
  • ovarian tumours as described herein in a subject in need of such treatment, comprising administering to said subject a therapeutically or prophylactically effective amount of Cystatin-C or fragments thereof;
  • Said ovarian tumour may be chosen from epithelial carcinoma, sex cord carcinoma, germ cell carcinoma, infiltrated metastatic carcinomas, cystadenoma, fibroma, thecoma, cystadenofibroma, mature teratoma, endometriosis, follicular cyst, abces, struma ovarii, Leydig cell tumour, parasalpingeal cyst, hydrosalpinx, corpus luteum cyst, heamorragic cyst, tissue with calcifications NOS, necrotic tumour NOS or combinations thereof.
  • said ovarian tumour is malignant, and said treatment is used to invoke regression, or growth inhibition of the malignant tumour.
  • the present invention thus unequivocally shows that using Cystatin-C as an additional diagnostic marker detectable in a blood or serum sample is beneficial for classifying ovarian tumours. This is unexpected due to the conflicting reports in the prior art (Weifang et al., 2010, PLoS One vol. 5 issue 1 1 and Kolwijck et al., 2010, Journal of Cancer Research and Clinical Oncology, 136: 771-778).
  • Figure 1 illustrates the mRNA and amino acid sequence of Cystatin-C (SEQ ID N0.1 and 2 respectively),
  • the peptides detected by the MASSterclassTM technology are depicted as SEQ ID No's 3 and 4 and are depicted as bold underlined or bold italic in the amino acid sequence.
  • biomarker is widespread in the art and may broadly denote a biological molecule and/or a detectable portion thereof whose qualitative and/or quantitative evaluation in a subject is predictive or informative (e.g., predictive, diagnostic and/or prognostic) with respect to one or more aspects of the subject's phenotype and/or genotype, such as, for example, with respect to the status of the subject as to a given disease or condition.
  • ovarian cancer refers to any cancerous growth arising from different parts of the ovary, or from infiltrated metastatic cancers originating from diverse origins. Most ovarian cancers are derived from epithelial tissue (epithelial ovarian cancer or "EOC"), Fallopian tubes, egg cells (germ cell tumours), sex cord, or stromal tissue. Alternatively, metastatic ovarian cancers, are cancers that have infiltrated from other tissue (e.g. breast cancer, endometrial cancer, lymphomas etc.) .
  • ovarian cyst ovarian mass
  • ovarian mass ovarian mass
  • ovarian cancer ovarian cancer
  • An ovarian cyst or mass can be considered to be benign if belonging to the group consisting of cystadenoma, fibroma, thecoma, cystadenofibroma, mature teratoma, endometriosis, follicular cyst, abces, struma ovarii, Leydig cell tumour, parasalpingeal cyst, hydrosalpinx, corpus luteum cyst, heamorragic cyst, tissue with calcifications NOS, necrotic tumour NOS or combinations thereof.
  • An ovarian cyst can be considered to be malignant if belonging to the group consisting of epithelial carcinoma, sex cord carcinoma, germ cell carcinoma or combinations thereof.
  • ovarian cyst encompasses any growth or mass formed on the ovaries or in the pelvis, including ovarian tumours, epithelial ovarian carcinoma (EOC), sex cord carcinoma, germ cell carcinoma, cystadenoma, fibroma, thecoma, cystadenofibroma, mature teratoma, endometriosis, follicular cyst, abces, struma ovarii, Leydig cell tumour, parasalpingeal cyst, hydrosalpinx, corpus luteum cyst, heamorragic cyst, tissue with calcifications NOS, necrotic tumour NOS or combinations thereof.
  • EOC epithelial ovarian carcinoma
  • sex cord carcinoma germ cell carcinoma
  • cystadenoma fibroma
  • fibroma thecoma
  • cystadenofibroma mature teratoma
  • endometriosis follicular cyst
  • abces struma ovarii
  • Leydig cell tumour parasalpingeal cyst
  • Signs and symptoms of ovarian cysts may include loss of appetite, indigestion, nausea, excessive gas and a bloated, full feeling, unexplained weight gain or an increased waist size, swelling in the abdomen, which can cause shortness of breath, pain in the lower abdomen, changes in bowel or bladder habits, such as constipation, diarrhoea or needing to pass urine more often, lower back pain, pain during sex, abnormal vaginal bleeding, etc.
  • the epithelial ovarian carcinoma can be selected from the group consisting of serous epithelial carcinoma, endometroid epithelial carcinoma, mucinous epithelial carcinoma, clear cell epithelial carcinoma, Brenner epithelial carcinoma, carcinosarcoma, undifferentiated epithelial carcinoma or combinations thereof.
  • the sex cord carcinoma can be selected from the group consisting of granulosa sex cord carcinoma, sertoli-leydig sex cord carcinoma, gyandroblastoma or combinations thereof.
  • the germ cell carcinoma can be selected from the group consisting of dysgerminoma, yolk sac carcinoma, embryonal carcinoma, choriocarcinoma, immature teratoma, or combinations thereof.
  • the cystadenoma can be selected from serous cystadenoma, endometrioid cystadenoma, mucinous cystadenoma, clear cell cystadenoma, Brenner cystadenoma or combinations thereof.
  • the fibroma can be selected from the group consisting of fibroma, thecoma, fribrothecoma or combinations thereof.
  • the cystadenofibroma can be selected from the group consisting of serous cystadenofibroma, mucinous cystadenofibroma, clear cell cystadenofibroma, Brenner cystadenofibroma or combinations thereof.
  • the staging of the ovarian cancers can be performed according to the FIGO staging of ovarian carcinomas (Benedet et al, 2000, International J Gynecologic & Obstetrics, 70: 209-262). An overview on how an ovarian cancers is classified as belonging to a particular stage is provided in Table 1.
  • Tumour limited to the ovaries ⁇ Tumour limited to one ovary; capsule intact, no tumour on ovarian surface; no malignant cells in ascites or peritoneal washings
  • IB Tumour limited to both ovaries; capsule intact, no tumour on ovarian surface;
  • IC Tumour limited to one or both ovaries with any of the following: capsule ruptured, tumour on ovarian surface, malignant cells in ascites or peritoneal washings
  • Tumour involves one or both ovaries with pelvic extension
  • Tumour involves one or both ovaries with microscopically confined peritoneal metasasis outside the pelvis and/or regional lymph node metastasis
  • Table 1 Overview of FIGO staging of ovarian cancers.
  • Early stage ovarian cancers comprise all stage I and stage IIA ovarian cancers.
  • Advanced stage ovarian cancers comprise stage MB, IIC and all stage III and IV ovarian cancers.
  • borderline tumour or Low Malignant Potential ovarian tumour or cancer (LMP) refers to tumours that are made up of low-grade cells and that are unlikely to spread. Borderline tumours are usually completely cured by surgery and rarely require further treatment.
  • LMP Low Malignant Potential ovarian tumour or cancer
  • RMI Risk of Maligancy Index
  • RMI U x M X CA125 wherein (U) is a score defining the ultrasound findings, (M) is the menopausal status, and CA125 is the serum level of CA125 (levels above 35U/ml are abnormal).
  • the Ultrasound findings are generally scored with one point (score) for the occurrence of each of the following: multi-locular cyst, evidence of solid areas, evidence of metastases, presence of ascites, and bilateral lesions.
  • women with a high risk of malignancy (>75% chance) will be referred to a cancer centre
  • women with a moderate risk of malignancy (20% chance) will be referred to a cancer unit/gynae-oncologist
  • the low risk subjects ( ⁇ 3% chance of obtaining a malignant cancer) will be referred to a gynae unit/oncologist.
  • the grading of the ovarian cancers can give an idea of how quickly it may develop.
  • Grading can be performed for instance with a three-tiered grading system according to Silverberg (Int J Gynecol Pathol 2000: 19: 7-15).
  • Grade 1 or low grade refers to cancer cells that are growing slowly, look quite similar to normal cells (are well differentiated) and are less likely to spread than high-grade cancers.
  • Grade 2 or moderate grade refers to cancer cells that look more abnormal and are growing slightly more quickly.
  • Grade 3 or high grade refers to cancer cells that are growing more quickly, look very abnormal or are poorly differentiated and are more likely to spread than low-grade cancers.
  • subject typically denotes humans, but may also encompass reference to non-human animals, preferably warm-blooded animals, more preferably mammals, such as, e.g., non-human primates, rodents, canines, felines, equines, ovines, porcines, and the like.
  • non-human animals preferably warm-blooded animals, more preferably mammals, such as, e.g., non-human primates, rodents, canines, felines, equines, ovines, porcines, and the like.
  • the terms “stratification” and “stratifying”, as used herein, encompass the process or result of discriminating, sorting or separating a population into more homogenous subpopulations according to specified criteria.
  • the term “risk stratification” or “stratifying subjects for the risk of indicates the grouping of patients in which a pelvic mass has been identified into different risk groups, reflecting their risk of having or developing a benign ovarian cyst versus a malignant ovarian cancer, more preferably wherein said malignant ovarian cancer is an early stage malignant ovarian cancer or a borderline (LMP) tumour.
  • LMP borderline
  • classification indicates establishing the malignancy of said tumour, i.e. whether said tumour is benign vs. malignant, preferably whether said tumour is a benign tumour vs. an early stage malignant cancer, and/or a borderline tumour with low malignant potential (LMP).
  • LMP low malignant potential
  • sample or “biological sample” as used herein include any biological specimen obtained from a subject.
  • Samples may include, without limitation, whole blood, plasma, serum, red blood cells, white blood cells (e.g., peripheral blood mononuclear cells), saliva, urine, stool ⁇ i.e., faeces), tears, sweat, sebum, nipple aspirate, ductal lavage, tumour exudates, synovial fluid, cerebrospinal fluid, lymph, fine needle aspirate, amniotic fluid, any other bodily fluid, cell lysates, cellular secretion products, inflammation fluid, semen and vaginal secretions.
  • Preferred samples may include ones comprising Cystatin-C proteins or fragments thereof in detectable quantities.
  • the sample may be whole blood or a fractional component thereof such as, e.g., plasma, serum, or a cell pellet.
  • the sample is readily obtainable by minimally invasive methods, allowing to remove or isolate said sample from the subject.
  • Samples may also include tissue samples and biopsies e.g. from the ovaries, tissue homogenates and the like.
  • the sample used to detect Cystatin-C levels is blood plasma.
  • the sample used to detect Cystatin-C levels is serum.
  • plasma defines the colourless watery fluid of the blood that contains no cells, but in which the blood cells (erythrocytes, leukocytes, thrombocytes, etc.) are suspended, containing nutrients, sugars, proteins, minerals, enzymes, etc.
  • serum defines the colourless watery fluid of the blood that contains no cells and no fibrinogens, but in which the blood cells (erythrocytes, leukocytes, thrombocytes, etc.) are suspended, containing nutrients, sugars, minerals, enzymes, proteins with the exception of those used in blood clotting, etc.
  • a molecule or analyte such as a protein, polypeptide or peptide, or a group of two or more molecules or analytes such as two or more proteins, polypeptides or peptides, is "measured" in a sample when the quantity of said molecule or analyte or of said group of molecules or analytes is detected or determined in the sample, preferably substantially to the exclusion of other molecules and analytes.
  • Quantity is synonymous and generally well-understood in the art.
  • the terms as used herein may particularly refer to an absolute quantification of a molecule or an analyte in a sample, or to a relative quantification of a molecule or analyte in a sample, i.e., relative to another value such as relative to a reference value as taught herein, or to a range of values indicating a base-line expression of the biomarker. These values or ranges can be obtained from a single patient or from a group of patients.
  • An absolute quantity of a molecule or analyte in a sample may be advantageously expressed as weight or as molar amount, or more commonly as a concentration, e.g., weight per volume or mol per volume.
  • a relative quantity of a molecule or analyte in a sample may be advantageously expressed as an increase or decrease or as a fold-increase or fold-decrease relative to another value, such as relative to a reference value as taught herein.
  • first and second parameters e.g., first and second quantities
  • a measurement method can produce quantifiable readouts (such as, e.g., signal intensities) for said first and second parameters, wherein said readouts are a function of the value of said parameters, and wherein said readouts can be directly compared to produce a relative value for the first parameter vs. the second parameter, without the actual need to first convert the readouts to absolute values of the respective parameters.
  • said "value” or “risk value” can also be presented using the risk stratification tool of the invention, wherein the quantities, measurements or scores for the biomarker(s) and parameter(s) may each be modulated by an appropriate weighing factor and added up to yield a single value, which can then be suitably compared with a corresponding reference value obtained accordingly.
  • said risk stratification tool is based on the concentrations of Cystatin-C or fragments thereof, optionally compensated by the measured eGfr value, and the concentration of HE4 and/or CA125, optionally combined with clinical parameters, risk factors, or other biomarkers.
  • the risk stratification tool is an equation, that yields a certain value, which can be projected on a scale, indicative for the risk of malignancy of the ovarian mass in a subject.
  • the clinical and morphological analysis of the pelvic mass through e.g. Ultrasound (US), Magnetic Resonance Imaging (MRI), Computed Tomography (CT), or any combination of said imaging methods can be used to calculate the value.
  • the concentration of one or more other biomarkers in the sample from said subject can be added to the risk stratification tool to obtain a value
  • Certain other risk factors such as genetic predisposition such as BRCA-gene mutations such as mutations in the BRCA-1 , BRCA-2, BRCA-3, and/or BRCA-X gene(s), age, diet, obesity, reproductive history, menopausal status, gynaecological surgery such as tubal ligation and hysterectomy, hormonal (replacement) therapy, smoking and alcohol use thereof can further be added to the risk stratification tool to obtain a value.
  • Cystatin-C corresponds to the protein commonly known as Cystatin-C, also known as “Cystatin 3" or previously "gamma trace” i.e. the proteins and polypeptides commonly known under these designations in the art, such as those proteins encoded and encoded by the CST3 gene.
  • the terms encompass such proteins and polypeptides of any organism where found, and particularly of animals, preferably vertebrates, more preferably mammals, including humans and non-human mammals, even more preferably of humans.
  • the terms particularly encompass such proteins and polypeptides with a native sequence, i.e., ones of which the primary sequence is the same as that of Cystatin-C found in or derived from nature.
  • Cystatin-C may differ between different species due to genetic divergence between such species. Moreover, the native sequences of Cystatin-C may differ between or within different individuals of the same species due to normal genetic diversity (variation) within a given species. Also, the native sequences of Cystatin-C may differ between or even within different individuals of the same species due to post-transcriptional or post-translational modifications. Accordingly, all Cystatin-C sequences found in or derived from nature are considered "native".
  • the terms encompass Cystatin-C proteins and polypeptides when forming a part of a living organism, organ, tissue or cell, when forming a part of a biological sample, as well as when at least partly isolated from such sources. The terms also encompass proteins and polypeptides when produced by recombinant or synthetic means.
  • Cystatin-C includes, without limitation, human Cystatin-C encoded by the mRNA as annotated under NCBI Genbank (http://www.ncbi.nlm.nih.gov/) accession number NM_000099 (sequence version 2) as depiceted in Figure 1 (SEQ ID NO: 1 ).
  • Said mRNA sequence encodes a polypeptide comprising 146 amino acids ( Figure 1 ) and annotated under NCBI Genbank (http://www.ncbi.nlm.nih.gov/) accession number NP_000090 (sequence version 1 ) (SEQ ID NO: 2).
  • a skilled person can also appreciate that said sequences are of precursor of Cystatin-C and may include parts which are processed away from mature Cystatin-C.
  • the circulating Cystatin-C e.g. a released or secreted form circulating in the blood, e.g. plasma or serum, may be detected, as opposed to the cell-bound or cell-confined Cystatin-C protein.
  • Cystatin-C may thus also encompass fragments of Cystatin-C.
  • the reference herein to measuring Cystatin-C, or to measuring the quantity of Cystatin-C may encompass measuring the Cystatin-C protein or polypeptide, such as, e.g., measuring the mature and/or the processed soluble/secreted form (e.g. plasma circulating form) of Cystatin- C and/or measuring one or more fragments thereof.
  • Cystatin-C and/or one or more fragments thereof may be measured collectively, such that the measured quantity corresponds to the sum amounts of the collectively measured species, by for example using a binding molecule that binds at the C-terminal end of Cystatin-C.
  • Cystatin- C and/or one or more fragments thereof may be measured each individually.
  • said fragment of Cystatin-C is a serum and/or plasma circulating form of Cystatin-C.
  • the expression "serum and/or plasma circulating form of Cystatin-C" or shortly “circulating form” encompasses all Cystatin-C proteins or fragments thereof that circulate in the serum and/or plasma, i.e., are not cell- or membrane-bound. Without wanting to be bound by any theory, such circulating forms can be derived from the full-length Cystatin-C protein through natural processing, or can be resulting from known degradation processes occurring in said sample.
  • the circulating form can also be the full-length Cystatin-C protein, which is found to be circulating in the serum and/or plasma.
  • Said "circulating form” can thus be any Cystatin-C protein or any processed soluble form of Cystatin-C or fragments of either one, that is circulating in the sample, i.e. which is not bound to a cell- or membrane fraction of said sample.
  • Cystatin-C peptide is known to be an angiogenesis inhibitor and is known to inhibit cathepsin- L, which is responsible for the cleavage of the LG-3 domain from the Perlecan/endorepellin angiogenesis inhibitor.
  • the term "creatinin” corresponds to a break-down product of creatine phosphate in muscle and is a cyclic derivative of creatine. Creatinine is filtered out of the blood by the kidneys (glomerular filtration and proximal tubular secretion). If the filtering of the kidney is deficient however, creatinine blood levels rise and hence creatinine levels in blood and urine are often used to calculate the creatinine clearance (CrCI), which reflects the glomerular filtration rate (GFR).
  • eGfr estimated glomerular filtration rate
  • cystatin-C is a well known filtration marker and that an aberrantly high or abnormally low kidney function could hamper the performance of the marker in predicting or prognosis of ovarian cancer. For this reason, logistic regression models can be used taking into account the eGfr to compensate for the filtration function and to "normalise”, or “correct” the cystatin-C levels, thereby increasing its performance in the diagnosis of ovarian cancer.
  • CA125 also known as mucin 16 (MUC16), FLJ14303, refers to peptides commonly known under these designations in the art, as exemplarily annotated under Genbank accession number NP_078966 (sequence version 2).
  • ⁇ 4 also known as WAP four-disulfide core domain 2 (WFDC2), WAP5; EDDM4; MGC57529; dJ461 P17.6; refers to peptides commonly known under these designations in the art.
  • WFDC2 WAP four-disulfide core domain 2
  • HE4a alternative sequence of the HE4 protein, termed "HE4a” was published by Hellstrom et al., 2003 (Cancer Res. 63 (13), 3695-3700) and annotated in Genbank as AY212888.
  • MSLN mesothelin
  • SMRP refers to peptides commonly known under these designations in the art, as exemplarily annotated under Genbank accession number NP_005814 (sequence version 2).
  • CA72-4 also known as inter alia tumour-associated glycoprotein (TAG-72), refers to peptides commonly known under these designations in the art.
  • osteopontin or ⁇
  • bone sialoprotein I also known as bone sialoprotein I (BSPI, BSP-1 or BNSP), early T-lymphocyte activation (ETA-1 ), secreted phosphoprotein 1 (SPP1 ) or MGC1 10940
  • BSPI bone sialoprotein I
  • ETA-1 early T-lymphocyte activation
  • SPP1 secreted phosphoprotein 1
  • MGC1 10940 refers to peptides commonly known under these designations in the art, as exemplarily annotated under Genbank accession number NP_001035147 (sequence version 1 ).
  • inhibitor refers to a dimer consisting of an alpha subunit and a beta subunit.
  • inhibitor, alpha or “INHA” refers to peptides commonly known under these designations in the art, as exemplarily annotated under Genbank accession number NP_002182 (sequence version 1 ).
  • inhibitor, beta A or “INHBA”, also known as EDF or FRP, refers to peptides commonly known under these designations in the art, as exemplarily annotated under Genbank accession number NP_000007 (sequence version 13).
  • inhibitor, beta B or "INHBB”, also known as MGC157939 refers to peptides commonly known under these designations in the art, as exemplarily annotated under Genbank accession number NP_002184 (sequence version 2).
  • inhibitor, beta C or "INHBC”, also known as INHB refers to peptides commonly known under these designations in the art, as exemplarily annotated under Genbank accession number NP_005529 (sequence version 1 ).
  • any protein, polypeptide or peptide encompasses such from any organism where found, and particularly preferably from animals, preferably vertebrates, more preferably mammals, including humans and non-human mammals, even more preferably from humans.
  • any protein, polypeptide or peptide and fragments thereof may generally also encompass modified forms of said protein, polypeptide or peptide and fragments such as bearing post-expression modifications including, for example, phosphorylation, glycosylation, lipidation, methylation, cysteinylation, sulphonation, glutathionylation, acetylation, oxidation of methionine to methionine sulphoxide or methionine sulphone, and the like.
  • Cystatin-C and fragments thereof, or other biomarkers as employed herein and fragments thereof may be human, i.e., their primary sequence may be the same as a corresponding primary sequence of or present in a naturally occurring human peptides, polypeptides or proteins.
  • the qualifier "human” in this connection relates to the primary sequence of the respective proteins, polypeptides, peptides or fragments, rather than to their origin or source.
  • proteins, polypeptides, peptides or fragments may be present in or isolated from samples of human subjects or may be obtained by other means (e.g., by recombinant expression, cell-free translation or non-biological peptide synthesis).
  • fragment of a protein, polypeptide or peptide generally refers to N-terminally and/or C-terminally deleted or truncated forms of said protein, polypeptide or peptide.
  • the term encompasses fragments arising by any mechanism, such as, without limitation, by alternative translation, exo- and/or endo-proteolysis and/or degradation of said protein or polypeptide, such as, for example, in vivo or in vitro, such as, for example, by physical, chemical and/or enzymatic proteolysis.
  • a fragment of a protein, polypeptide or peptide may represent at least about 5%, or at least about 10%, e.g., ⁇ 20%, > 30% or > 40%, such as > 50%, e.g., ⁇ 60%, > 70% or > 80%, or even > 90% or > 95% of the amino acid sequence of said protein, polypeptide or peptide.
  • a fragment may include a sequence of > 5 consecutive amino acids, or > 10 consecutive amino acids, or > 20 consecutive amino acids, or > 30 consecutive amino acids, e.g., >40 consecutive amino acids, such as for example > 50 consecutive amino acids, e.g., ⁇ 60, > 70, > 80, > 90, > 100, > 200, > 300, > 400, > 500 or > 600 consecutive amino acids of the corresponding full length protein.
  • a fragment may be N-terminally and/or C-terminally truncated by between 1 and about 20 amino acids, such as, e.g., by between 1 and about 15 amino acids, or by between 1 and about 10 amino acids, or by between 1 and about 5 amino acids, compared to the corresponding mature, full-length protein or its soluble, serum or plasma circulating form.
  • fragments of a given protein, polypeptide or peptide may be achieved by in vitro proteolysis of said protein, polypeptide or peptide to obtain advantageously detectable peptide(s) from a sample.
  • proteolysis may be effected by suitable physical, chemical and/or enzymatic agents, e.g., proteinases, preferably endoproteinases, i.e., protease cleaving internally within a protein, polypeptide or peptide chain.
  • suitable endoproteinases includes serine proteinases (EC 3.4.21 ), threonine proteinases (EC 3.4.25), cysteine proteinases (EC 3.4.22), aspartic acid proteinases (EC 3.4.23), metalloproteinases (EC 3.4.24) and glutamic acid proteinases.
  • Exemplary non-limiting endoproteinases include trypsin, chymotrypsin, elastase, Lysobacter enzymogenes endoproteinase Lys-C, Staphylococcus aureus endoproteinase Glu-C (endopeptidase V8) or Clostridium histolyticum endoproteinase Arg-C (clostripain). Further known or yet to be identified enzymes may be used; a skilled person can choose suitable protease(s) on the basis of their cleavage specificity and frequency to achieve desired peptide forms.
  • the proteolysis may be effected by endopeptidases of the trypsin type (EC 3.4.21 .4), preferably trypsin, such as, without limitation, preparations of trypsin from bovine pancreas, human pancreas, porcine pancreas, recombinant trypsin, Lys-acetylated trypsin, trypsin in solution, trypsin immobilised to a solid support, etc. Trypsin is particularly useful, inter alia due to high specificity and efficiency of cleavage.
  • the invention also contemplates the use of any trypsin-like protease, i.e., with a similar specificity to that of trypsin.
  • chemical reagents may be used for proteolysis.
  • CNBr can cleave at Met
  • BNPS-skatole can cleave at Trp.
  • the conditions for treatment e.g., protein concentration, enzyme or chemical reagent concentration, pH, buffer, temperature, time, can be determined by the skilled person depending on the enzyme or chemical reagent employed.
  • isolated with reference to a particular component (such as for instance, a protein, polypeptide, peptide or fragment thereof) generally denotes that such component exists in separation from - for example, has been separated from or prepared in separation from - one or more other components of its natural environment.
  • a particular component such as for instance, a protein, polypeptide, peptide or fragment thereof
  • an isolated human or animal protein, polypeptide, peptide or fragment exists in separation from a human or animal body where it occurs naturally.
  • isolated may preferably also encompass the qualifier "purified".
  • purified with reference to protein(s), polypeptide(s), peptide(s) and/or fragment(s) thereof does not require absolute purity. Instead, it denotes that such protein(s), polypeptide(s), peptide(s) and/or fragment(s) is (are) in a discrete environment in which their abundance (conveniently expressed in terms of mass or weight or concentration) relative to other proteins is greater than in a biological sample.
  • a discrete environment denotes a single medium, such as for example a single solution, gel, precipitate, lyophilisate, etc.
  • Purified peptides, polypeptides or fragments may be obtained by known methods including, for example, laboratory or recombinant synthesis, chromatography, preparative electrophoresis, centrifugation, precipitation, affinity purification, etc.
  • Purified protein(s), polypeptide(s), peptide(s) and/or fragment(s) may preferably constitute by weight > 10%, more preferably > 50%, such as > 60%, yet more preferably > 70%, such as > 80%, and still more preferably > 90%, such as > 95%, > 96%, > 97%, > 98%, > 99% or even 100%, of the protein content of the discrete environment.
  • Protein content may be determined, e.g., by the Lowry method (Lowry et al. 1951 . J Biol Chem 193: 265), optionally as described by Hartree 1972 (Anal Biochem 48: 422-427).
  • purity of peptides or polypeptides may be determined by SDS-PAGE under reducing or non-reducing conditions using Coomassie blue or, preferably, silver stain.
  • label refers to any atom, molecule, moiety or biomolecule that can be used to provide a detectable and preferably quantifiable read-out or property, and that can be attached to or made part of an entity of interest, such as a peptide or polypeptide or a specific- binding agent. Labels may be suitably detectable by mass spectrometric, spectroscopic, optical, colorimetric, magnetic, photochemical, biochemical, immunochemical or chemical means.
  • Labels include without limitation dyes; radiolabels such as 32 P, 33 P, 35 S, 125 l, 131 l; electron-dense reagents; enzymes (e.g. , horse-radish phosphatise or alkaline phosphatase as commonly used in immunoassays); binding moieties such as biotin-streptavidin; haptens such as digoxigenin; luminogenic, phosphorescent or fluorogenic moieties; mass tags; and fluorescent dyes alone or in combination with moieties that can suppress or shift emission spectra by fluorescence resonance energy transfer (FRET).
  • the label may be a mass-altering label.
  • a mass-altering label may involve the presence of a distinct stable isotope in one or more amino acids of the peptide visa-vis its corresponding non-labelled peptide.
  • Mass-labelled peptides are particularly useful as positive controls, standards and calibrators in mass spectrometry applications.
  • peptides including one or more distinct isotopes are chemically alike, separate chromatographically and electrophoretically in the same manner and also ionise and fragment in the same way.
  • such peptides and optionally select fragmentation ions thereof will display distinguishable m/z ratios and can thus be discriminated.
  • pairs of distinguishable stable isotopes include H and D, 12 C and 13 C, 14 N and 15 N or 16 0 and 18 0.
  • peptides and proteins of biological samples analysed in the present invention may substantially only contain common isotopes having high prevalence in nature, such as for example H, 12 C, 14 N and 16 0.
  • the mass- labelled peptide may be labelled with one or more uncommon isotopes having low prevalence in nature, such as for instance D, 13 C, 15 N and/or 18 0. It is also conceivable that in cases where the peptides or proteins of a biological sample would include one or more uncommon isotopes, the mass-labelled peptide may comprise the respective common isotope(s).
  • Isotopically-labelled synthetic peptides may be obtained inter alia by synthesising or recombinantly producing such peptides using one or more isotopically-labelled amino acid substrates, or by chemically or enzymatically modifying unlabelled peptides to introduce thereto one or more distinct isotopes.
  • any amino acid of which deuterated or 15 N- or 13 C-containing forms exist may be considered for synthesis or recombinant production of labelled peptides.
  • a peptide may be treated with trypsin in H 2 16 0 or H 2 18 0, leading to incorporation of two oxygens ( 16 0 or 18 0, respectively) at the COOH-termini of said peptide (e.g., US 2006/105415).
  • Cystatin-C and isolated fragments thereof as taught herein, optionally comprising a detectable label, as (positive) controls, standards or calibrators in qualitative or quantitative detection assays (measurement methods) of Cystatin- C or creatinine, and particularly in such methods for the classification of an ovarian tumour in a subject as taught herein.
  • the proteins, polypeptides or peptides may be supplied in any form, inter alia as precipitate, vacuum-dried, lyophilisate, in solution as liquid or frozen, or covalently or non-covalently immobilised on solid phase, such as for example, on solid chromatographic matrix or on glass or plastic or other suitable surfaces (e.g., as a part of peptide arrays and microarrays).
  • the peptides may be readily prepared, for example, isolated from natural sources, or prepared recombinantly or synthetically.
  • binding agents capable of specifically binding to any one or more of the isolated fragments of Cystatin-C as taught herein.
  • a Cystatin-C binding molecule according to the invention is any substance that binds specifically to Cystatin-C.
  • binding agents capable of specifically binding to only one of isolated fragments of Cystatin-C as taught herein. Binding agents as intended throughout this specification may include inter alia an antibody, aptamer, photoaptamer, protein, peptide, a lipid, a carbohydrate, a nucleic acid, peptide-nucleic acid peptidomimetic, a small molecule or small organic molecule.
  • Cystatin-C binding molecule can be natural or synthetic compound, including, for example, synthetic small molecule, compound contained in extracts of animal, plant, bacterial or fungal cells, as well as conditioned medium from such cells.
  • Cystatin-C binding molecule can be an engineered protein having binding sites for Cystatin-C.
  • a binding agent may be capable of binding the serum and/or plasma circulating form and the cell-bound or retained from of Cystatin-C or creatinine.
  • a binding agent may be capable of specifically binding or detecting the serum and/or plasma circulating form of Cystatin-C or creatinine.
  • said binding molecule binds specifically to either Cystatin-C or fragments of either one of those, with an affinity better than 10 "6 M.
  • a suitable Cystatin-C binding molecule can be determined from its binding with a standard sample of Cystatin-C. Methods for determining the binding between binding molecules and Cystatin-C are known in the art.
  • the term antibody includes, but is not limited to, polyclonal antibodies, monoclonal antibodies, humanised or chimeric antibodies, engineered antibodies, and biologically functional antibody fragments (e.g. scFv, nanobodies, Fv, etc) sufficient for binding of the antibody fragment to the protein.
  • Such antibody may be commercially available antibody against Cystatin-C, such as, for example, a mouse, rat, human or humanised monoclonal antibody.
  • the Cystatin-C binding molecule is labelled with a tag that permits detection with another agent (e.g. with a probe binding partner).
  • tags can be, for example, biotin, streptavidin, his-tag, myc tag, maltose, maltose binding protein or any other kind of tag known in the art that has a binding partner.
  • Example of associations which can be utilised in the probe:binding partner arrangement may be any, and includes, for example biotin:streptavidin, his-tag:metal ion (e.g. Ni 2+ ), maltose:maltose binding protein.
  • kits may be in various forms, e.g., lyophilised, free in solution or immobilised on a solid phase. They may be, e.g., provided in a multi-well plate or as an array or microarray, or they may be packaged separately and/or individually. The may be suitably labelled as taught herein. Said kits may be particularly suitable for performing the assay methods of the invention, such as, e.g., immunoassays, ELISA assays, mass spectrometry assays, and the like.
  • binding agent binds to one or more desired molecules or analytes, such as to one or more proteins, polypeptides or peptides of interest or fragments thereof substantially to the exclusion of other molecules which are random or unrelated, and optionally substantially to the exclusion of other molecules that are structurally related.
  • binding agent binds to one or more desired molecules or analytes, such as to one or more proteins, polypeptides or peptides of interest or fragments thereof substantially to the exclusion of other molecules which are random or unrelated, and optionally substantially to the exclusion of other molecules that are structurally related.
  • specifically bind does not necessarily require that an agent binds exclusively to its intended target(s).
  • an agent may be said to specifically bind to protein(s) polypeptide(s), peptide(s) and/or fragment(s) thereof of interest if its affinity for such intended target(s) under the conditions of binding is at least about 2-fold greater, preferably at least about 5-fold greater, more preferably at least about 10-fold greater, yet more preferably at least about 25-fold greater, still more preferably at least about 50-fold greater, and even more preferably at least about 100-fold or more greater, than its affinity for a non-target molecule.
  • antibody is used in its broadest sense and generally refers to any immunologic binding agent.
  • the term specifically encompasses intact monoclonal antibodies, polyclonal antibodies, multivalent (e.g., 2-, 3- or more-valent) and/or multi-specific antibodies (e.g., bi- or more-specific antibodies) formed from at least two intact antibodies, and antibody fragments insofar they exhibit the desired biological activity (particularly, ability to specifically bind an antigen of interest), as well as multivalent and/or multi-specific composites of such fragments.
  • antibody is not only inclusive of antibodies generated by methods comprising immunisation, but also includes any polypeptide, e.g., a recombinantly expressed polypeptide, which is made to encompass at least one complementarity-determining region (CDR) capable of specifically binding to an epitope on an antigen of interest. Hence, the term applies to such molecules regardless whether they are produced in vitro or in vivo.
  • CDR complementarity-determining region
  • An antibody may be any of IgA, IgD, IgE, IgG and IgM classes, and preferably IgG class antibody.
  • An antibody may be a polyclonal antibody, e.g., an antiserum or immunoglobulins purified there from (e.g., affinity-purified).
  • An antibody may be a monoclonal antibody or a mixture of monoclonal antibodies.
  • Monoclonal antibodies can target a particular antigen or a particular epitope within an antigen with greater selectivity and reproducibility. By means of example and not limitation, monoclonal antibodies may be made by the hybridoma method first described by Kohler et al.
  • Monoclonal antibodies may also be isolated from phage antibody libraries using techniques as described by Clackson et al. 1991 (Nature 352: 624- 628) and Marks et al. 1991 (J Mol Biol 222: 581-597), for example.
  • Antibody binding agents may be antibody fragments.
  • Antibody fragments comprise a portion of an intact antibody, comprising the antigen-binding or variable region thereof.
  • Examples of antibody fragments include Fab, Fab', F(ab')2, Fv and scFv fragments; diabodies; linear antibodies; single-chain antibody molecules; and multivalent and/or multispecific antibodies formed from antibody fragment(s), e.g., dibodies, tribodies, and multibodies.
  • the above designations Fab, Fab', F(ab')2, Fv, scFv etc. are intended to have their art-established meaning.
  • antibody includes antibodies originating from or comprising one or more portions derived from any animal species, preferably vertebrate species, including, e.g., birds and mammals.
  • the antibodies may be chicken, turkey, goose, duck, guinea fowl, quail or pheasant.
  • the antibodies may be human, murine (e.g., mouse, rat, etc.), donkey, rabbit, goat, sheep, guinea pig, camel (e.g., Camelus bactrianus and Camelus dromaderius), lama (e.g., Lama paccos, Lama glama or Lama vicugna) or horse.
  • an antibody can include one or more amino acid deletions, additions and/or substitutions (e.g., conservative substitutions), insofar such alterations preserve its binding of the respective antigen.
  • An antibody may also include one or more native or artificial modifications of its constituent amino acid residues (e.g., glycosylation, etc.).
  • aptamer refers to single-stranded or double-stranded oligo-DNA, oligo-RNA or oligo-DNA/RNA or any analogue thereof that can specifically bind to a target molecule such as a peptide.
  • aptamers can display fairly high specificity and affinity (e.g., K A in the order 1x10 9 M "1 ) for their targets.
  • photoaptamer refers to an aptamer that contains one or more photoreactive functional groups that can covalently bind to or crosslink with a target molecule.
  • peptidomimetic refers to a non-peptide agent that is a topological analogue of a corresponding peptide.
  • small molecule refers to compounds, preferably organic compounds, with a size comparable to those organic molecules generally used in pharmaceuticals.
  • Preferred small organic molecules range in size up to about 5000 Da, e.g., up to about 4000, preferably up to 3000 Da, more preferably up to 2000 Da, even more preferably up to about 1000 Da, e.g., up to about 900, 800, 700, 600 or up to about 500 Da.
  • the animals to be immunised may include any animal species, preferably warm-blooded species, more preferably vertebrate species, including, e.g., birds and mammals.
  • the antibodies may be chicken, turkey, goose, duck, guinea fowl, quail or pheasant.
  • the antibodies may be human, murine (e.g., mouse, rat, etc.), donkey, rabbit, goat, sheep, guinea pig, camel, lama or horse.
  • presenting carrier or “carrier” generally denotes an immunogenic molecule which, when bound to a second molecule, augments immune responses to the latter, usually through the provision of additional T cell epitopes.
  • the presenting carrier may be a (poly)peptidic structure or a non-peptidic structure, such as inter alia glycans, polyethylene glycols, peptide mimetics, synthetic polymers, etc.
  • Exemplary non- limiting carriers include human Hepatitis B virus core protein, multiple C3d domains, tetanus toxin fragment C or yeast Ty particles.
  • Immune sera obtained or obtainable by immunisation as taught herein may be particularly useful for generating antibody reagents that specifically bind to one or more of the herein disclosed fragments of Cystatin-C or creatinine.
  • Such methods may be based on subtracting or removing binding agents which cross-react or cross-bind the non-desired Cystatin-C molecules under (b). Such subtraction may be readily performed as known in the art by a variety of affinity separation methods, such as affinity chromatography, affinity solid phase extraction, affinity magnetic extraction, etc.
  • any existing, available or conventional separation, detection and quantification methods can be used herein to measure the presence or absence (e.g., readout being present vs. absent; or detectable amount vs. undetectable amount) and/or quantity (e.g., readout being an absolute or relative quantity, such as, for example, absolute or relative concentration) of Cystatin-C and/or fragments thereof and optionally of CA125, HE4 and/or the one or more other biomarkers or fragments thereof in samples (any molecules or analytes of interest to be so-measured in samples, including Cystatin-C and fragments thereof, may be herein below referred to collectively as biomarkers).
  • such methods may include immunoassay methods, mass spectrometry analysis methods, or chromatography methods, or combinations thereof.
  • immunoassay generally refers to methods known as such for detecting one or more molecules or analytes of interest in a sample, wherein specificity of an immunoassay for the molecule(s) or analyte(s) of interest is conferred by specific binding between a specific- binding agent, commonly an antibody, and the molecule(s) or analyte(s) of interest.
  • Immunoassay technologies include without limitation direct ELISA (enzyme-linked immunosorbent assay), indirect ELISA, sandwich ELISA, competitive ELISA, multiplex ELISA, radioimmunoassay (RIA), ELISPOT technologies, and other similar techniques known in the art. Principles of these immunoassay methods are known in the art, for example John R. Crowther, "The ELISA Guidebook", 1 st ed., Humana Press 2000, ISBN 0896037282;
  • labelling in ELISA technologies is usually by enzyme (such as, e.g., horse-radish peroxidase) conjugation and the end-point is typically colorimetric, chemiluminescent or fluorescent, magnetic, piezo electric, pyroelectric and other.
  • enzyme such as, e.g., horse-radish peroxidase
  • Radioimmunoassay is a competition-based technique and involves mixing known quantities of radioactively-labelled (e.g., 125 l- or 131 l-labelled) target antigen with antibody to said antigen, then adding non-labelled or 'cold' antigen from a sample and measuring the amount of labelled antigen displaced (see, e.g., "An Introduction to Radioimmunoassay and Related Techniques", by Chard T, ed., Elsevier Science 1995, ISBN 0444821 198 for guidance).
  • radioactively-labelled e.g., 125 l- or 131 l-labelled
  • any mass spectrometric (MS) techniques that can obtain precise information on the mass of peptides, and preferably also on fragmentation and/or (partial) amino acid sequence of selected peptides (e.g., in tandem mass spectrometry, MS/MS; or in post source decay, TOF MS), are useful herein.
  • MS/MS mass spectrometric
  • Suitable peptide MS and MS/MS techniques and systems are well-known per se (see, e.g., Methods in Molecular Biology, vol. 146: "Mass Spectrometry of Proteins and Peptides", by Chapman, ed., Humana Press 2000, ISBN 089603609x; Biemann 1990. Methods Enzymol 193: 455-79; or Methods in Enzymology, vol.
  • MS arrangements, instruments and systems suitable for biomarker peptide analysis may include, without limitation, matrix-assisted laser desorption/ionisation time-of- flight (MALDI-TOF) MS; MALDI-TOF post-source-decay (PSD); MALDI-TOF/TOF; surface- enhanced laser desorption/ionization time-of-f light mass spectrometry (SELDI-TOF) MS; electrospray ionization mass spectrometry (ESI-MS); ESI-MS/MS; ESI-MS/(MS) n (n is an integer greater than zero); ESI 3D or linear (2D) ion trap MS; ESI triple quadrupole MS; ESI quadrupole orthogonal TOF (Q-TOF); ESI Fourier transform MS systems; desorption/ionization
  • MALDI-TOF matrix-assisted laser desorption/ionisation time-of- flight
  • PSD MALDI-TOF post-source-decay
  • MS/MS Peptide ion fragmentation in tandem MS
  • CID collision induced dissociation
  • Detection and quantification of biomarkers by mass spectrometry may involve multiple reaction monitoring (MRM), such as described among others by Kuhn et al. 2004 (Proteomics 4: 1 175-86).
  • MRM multiple reaction monitoring
  • MS peptide analysis methods may be advantageously combined with upstream peptide or protein separation or fractionation methods, such as for example with the chromatographic and other methods described herein below.
  • Chromatography can also be used for measuring biomarkers.
  • the term "chromatography” encompasses methods for separating chemical substances, referred to as such and vastly available in the art.
  • chromatography refers to a process in which a mixture of chemical substances (analytes) carried by a moving stream of liquid or gas (“mobile phase”) is separated into components as a result of differential distribution of the analytes, as they flow around or over a stationary liquid or solid phase (“stationary phase”), between said mobile phase and said stationary phase.
  • the stationary phase may be usually a finely divided solid, a sheet of filter material, or a thin film of a liquid on the surface of a solid, or the like.
  • Chromatography is also widely applicable for the separation of chemical compounds of biological origin, such as, e.g., amino acids, proteins, fragments of proteins or peptides, etc.
  • Chromatography as used herein may be preferably columnar (i.e., wherein the stationary phase is deposited or packed in a column), preferably liquid chromatography, and yet more preferably HPLC. While particulars of chromatography are well known in the art, for further guidance see, e.g., Meyer M., 1998, ISBN: 047198373X, and "Practical HPLC Methodology and Applications", Bidlingmeyer, B. A., John Wiley & Sons Inc., 1993.
  • Exemplary types of chromatography include, without limitation, high-performance liquid chromatography (HPLC), normal phase HPLC (NP-HPLC), reversed phase HPLC (RP-HPLC), ion exchange chromatography (I EC), such as cation or anion exchange chromatography, hydrophilic interaction chromatography (HILIC), hydrophobic interaction chromatography (HIC), size exclusion chromatography (SEC) including gel filtration chromatography or gel permeation chromatography, chromatofocusing, affinity chromatography such as immuno-affinity, immobilised metal affinity chromatography, and the like.
  • HPLC high-performance liquid chromatography
  • NP-HPLC normal phase HPLC
  • RP-HPLC reversed phase HPLC
  • I EC ion exchange chromatography
  • I EC ion exchange chromatography
  • HILIC hydrophilic interaction chromatography
  • HIC hydrophobic interaction chromatography
  • SEC size exclusion chromatography
  • gel filtration chromatography or gel permeation chromatography
  • Chromatography including single-, two- or more-dimensional chromatography, may be used as a peptide fractionation method in conjunction with a further peptide analysis method, such as for example, with a downstream mass spectrometry analysis as described elsewhere in this specification.
  • peptide or polypeptide separation, identification or quantification methods may be used, optionally in conjunction with any of the above described analysis methods, for measuring biomarkers in the present disclosure.
  • Such methods include, without limitation, chemical extraction partitioning, isoelectric focusing (IEF) including capillary isoelectric focusing (CIEF), capillary isotachophoresis (CITP), capillary electrochromatography (CEC), and the like, one-dimensional polyacrylamide gel electrophoresis (PAGE), two-dimensional polyacrylamide gel electrophoresis (2D-PAGE), capillary gel electrophoresis (CGE), capillary zone electrophoresis (CZE), micellar electrokinetic chromatography (MEKC), free flow electrophoresis (FFE), etc.
  • IEF isoelectric focusing
  • CITP capillary isotachophoresis
  • CEC capillary electrochromatography
  • PAGE polyacrylamide gel electrophoresis
  • 2D-PAGE two-dimensional polyacrylamide gel electrophore
  • the various aspects and embodiments taught herein may further rely on comparing the quantity of Cystatin-C or fragments thereof, as defined herein, measured in samples with reference values of the quantity of Cystatin-C, wherein said reference values represent a known classification of an ovarian tumour as taught herein.
  • distinct reference values may represent the classification of an ovarian tumour as malignant as taught herein vs. classification of an ovarian tumour as benign as taught herein.
  • distinct reference values may represent the classification of an ovarian tumour in said subject as belonging to a subclass of ovarian tumours.
  • Such comparison may generally include any means to determine the presence or absence of at least one difference and optionally of the size of such different between values or profiles being compared.
  • a comparison may include a visual inspection, an arithmetical or statistical comparison of measurements. Such statistical comparisons include, but are not limited to, applying a rule. If the values or biomarker profiles comprise at least one standard, the comparison to determine a difference in said values or biomarker profiles may also include measurements of these standards, such that measurements of the biomarker are correlated to measurements of the internal standards.
  • a reference value of the quantity of Cystatin-C for a particular classification of an ovarian tumour as taught herein may be established by determining the quantity of Cystatin-C in sample(s) from one individual or from a population of individuals characterised by said particular classification of an ovarian tumour (i.e., for whom said classification holds true). Such population may comprise without limitation > 2, > 10, > 100, or even several hundreds or more individuals.
  • reference values of the quantity of Cystatin-C for the classification of an ovarian tumour as malignant as taught herein vs.
  • classification of an ovarian tumour as benign as taught herein may be established by determining the quantity of Cystatin-C in sample(s) from one individual or from a population of individuals classified (e.g., based on other adequately conclusive means, such as, for example, clinical signs and symptoms, imaging, etc.) as, respectively, having a malignant or benign ovarian tumour.
  • reference value(s) as intended herein may convey absolute quantities of Cystatin-C.
  • the quantity of Cystatin-C in a sample from a tested subject may be determined directly relative to the reference value (e.g., in terms of increase or decrease, or fold-increase or fold-decrease).
  • this may allow to compare the quantity of Cystatin-C in the sample from the subject with the reference value (in other words to measure the relative quantity of Cystatin-C in the sample from the subject vis-a-vis the reference value) without the need to first determine the absolute quantity of Cystatin-C.
  • the expression level or presence of a biomarker in a sample of a patient may sometimes fluctuate, i.e.
  • the marker change precedes the change in symptoms and becomes a more sensitive measure than symptom change.
  • Therapeutic intervention can be initiated earlier and be more effective than waiting for deteriorating symptoms.
  • Measuring the Cystatin-C level of the same patient at different time points can in such a case thus enable the continuous monitoring of the status of the patient and can lead to prediction of worsening or improvement of the patient's condition with regard to a given disease or condition as taught herein.
  • a home or clinical test kit or device as indicated herein can be used for this continuous monitoring.
  • One or more reference values or ranges of Cystatin-C levels linked to a certain disease state can e.g. be determined beforehand or during the monitoring process over a certain period of time in said subject.
  • these reference values or ranges can be established through data sets of several patients with highly similar disease phenotypes, e.g. from subjects having a benign ovarian tumour or subjects having a malignant ovarian tumour.
  • a sudden deviation of the Cystatin-C levels from said reference value or range can predict the worsening of the condition of the patient (e.g. at home or in the clinic) before the (often severe) symptoms actually can be felt or observed.
  • the Cystatin-C value can be compensated or corrected by the measured eGfr value. Also disclosed is thus a method or algorithm for determining a significant change in the level of the Cystatin-C marker in a certain patient, which is indicative for change (worsening or improving) in clinical status.
  • the invention allows establishing the risk of having a malignant ovarian tumour as taught herein.
  • the Cystatin-C value can be compensated or corrected by the measured eGfr value. ln an embodiment the present methods may include a step of establishing such reference value(s).
  • kits and devices may include means for establishing a reference value of the quantity of Cystatin-C for a particular classification of an ovarian tumour in a subject as taught herein.
  • Such means may for example comprise one or more samples (e.g., separate or pooled samples) from one or more individuals characterised by said particular classification of an ovarian tumour.
  • the Cystatin-C value can be compensated or corrected by the measured eGfr value.
  • the various aspects and embodiments taught herein may further entail finding a deviation or no deviation between the quantity of Cystatin-C measured in a sample from a subject and a given reference value.
  • the Cystatin-C value can be compensated or corrected by the measured eGfr value.
  • a "deviation" of a first value from a second value may generally encompass any direction (e.g., increase: first value > second value; or decrease: first value ⁇ second value) and any extent of alteration.
  • a deviation may encompass a decrease in a first value by, without limitation, at least about 10% (about 0.9-fold or less), or by at least about 20% (about 0.8-fold or less), or by at least about 30% (about 0.7-fold or less), or by at least about 40% (about 0.6-fold or less), or by at least about 50% (about 0.5-fold or less), or by at least about 60% (about 0.4-fold or less), or by at least about 70% (about 0.3-fold or less), or by at least about 80% (about 0.2-fold or less), or by at least about 90% (about 0.1 -fold or less), relative to a second value with which a comparison is being made.
  • a deviation may encompass an increase of a first value by, without limitation, at least about 10% (about 1.1 -fold or more), or by at least about 20% (about 1.2-fold or more), or by at least about 30% (about 1.3-fold or more), or by at least about 40% (about 1.4-fold or more), or by at least about 50% (about 1.5-fold or more), or by at least about 60% (about 1.6- fold or more), or by at least about 70% (about 1.7-fold or more), or by at least about 80% (about 1.8-fold or more), or by at least about 90% (about 1.9-fold or more), or by at least about 100% (about 2-fold or more), or by at least about 150% (about 2.5-fold or more), or by at least about 200% (about 3-fold or more), or by at least about 500% (about 6-fold or more), or by at least about 700% (about 8-fold or more), or like, relative to a second value with which a comparison is being made.
  • a deviation may refer to a statistically significant observed alteration.
  • a deviation may refer to an observed alteration which falls outside of error margins of reference values in a given population (as expressed, for example, by standard deviation or standard error, or by a predetermined multiple thereof, e.g., ⁇ 1xSD or ⁇ 2xSD, or ⁇ 1xSE or ⁇ 2xSE).
  • Deviation may also refer to a value falling outside of a reference range defined by values in a given population (for example, outside of a range which comprises >40%, > 50%, >60%, >70%, >75% or >80% or >85% or >90% or >95% or even >100% of values in said population).
  • a deviation may be concluded if an observed alteration is beyond a given threshold or cut-off.
  • threshold or cut-off may be selected as generally known in the art to provide for a chosen sensitivity and/or specificity of the classification methods, e.g., sensitivity and/or specificity of at least 50%, or at least 60%, or at least 70%, or at least 80%, or at least 85%, or at least 90%, or at least 95%.
  • the Cystatin-C value can be compensated or corrected by the measured eGfr value.
  • the Cystatin-C value can be compensated or corrected by the measured eGfr value.
  • biomarker profile includes any set of data that represents the distinctive features or characteristics associated with a condition of interest, such as with a particular classification of an ovarian tumour as taught herein.
  • nucleic acid profiles such as for example genotypic profiles (sets of genotypic data that represents the genotype of one or more genes associated with a condition of interest), gene copy number profiles (sets of gene copy number data that represents the amplification or deletion of one or more genes associated with a condition of interest), gene expression profiles (sets of gene expression data that represents the mRNA levels of one or more genes associated with a condition of interest), DNA methylation profiles (sets of methylation data that represents the DNA methylation levels of one or more genes associated with a condition of interest), as well as protein, polypeptide or peptide profiles, such as for example protein expression profiles (sets of protein expression data that represents the levels of one or more proteins associated with a condition of interest), protein activation profiles (sets of data that represents the activation or inactivation of one or more proteins associated with a condition of interest), protein modification profiles (sets of data that represents the modification of one or more proteins associated with a condition of interest), protein cleavage profiles (sets of
  • Biomarker profiles may be created in a number of ways and may be the combination of measurable biomarkers or aspects of biomarkers using methods such as ratios, or other more complex association methods or algorithms (e.g., rule-based methods).
  • a biomarker profile comprises at least two measurements, where the measurements can correspond to the same or different biomarkers.
  • a biomarker profile may also comprise at least three, four, five, 10, 20, 30 or more measurements. In one embodiment, a biomarker profile comprises hundreds, or even thousands, of measurements.
  • distinct reference profiles may represent the classification (e.g., an abnormally elevated risk) of having a malignant ovarian tumour vs. the classification of having a benign ovarian tumour.
  • distinct reference profiles may represent the classification of an ovarian tumour in said subject as belonging to a subclass of ovarian tumours. Reference profiles used herein may be established according to known procedures previously employed for other biomarkers.
  • a reference profile of the quantity of Cystatin-C and the quantity of CA125, HE4 and/or one or more other biomarkers for a particular classification of an ovarian tumour as taught herein may be established by determining the profile in sample(s) from one individual or from a population of individuals characterised by said particular classification of an ovarian tumour (i.e., for whom said classification holds true).
  • population may comprise without limitation > 2, > 10, > 100, or even several hundreds or more individuals.
  • reference profiles for the classification of an ovarian tumour as malignant as taught herein vs. an ovarian tumour as benign as taught herein may be established by determining the biomarker profiles in sample(s) from one individual or from a population of individuals classified and/or diagnosed as having a malignant or benign ovarian tumour, respectively.
  • the present methods may include a step of establishing such reference profile(s).
  • the present kits and devices may include means for establishing a reference profile for a particular classification of an ovarian tumour as taught herein. Such means may for example comprise one or more samples (e.g., separate or pooled samples) from one or more individuals characterised by said particular classification of an ovarian tumour. Further, art-known multi-parameter analyses may be employed mutatis mutandis to determine deviations between groups of values and profiles generated there from (e.g., between sample and reference biomarker profiles).
  • biomarkers measured as taught herein that are each given a specific weight factor.
  • these biomarkers are Cystatin-C or a fragment thereof, optionally combined with other biomarkers and/or risk factors and/or clinical parameters.
  • said measurements, factors and/or parameters result in a numerical value which can be projected on a risk scale.
  • Said risk scale or risk stratification tool is indicative for the risk of having or developing ovarian cancer.
  • the risk scale is established by testing the same equation on populations of samples of subjects in which the ovarian mass has been previously correctly diagnosed or classified as being benign or malignant ovarian cancer, more preferably wherein said malignant ovarian cancer is an early stage malignant ovarian cancer or is a borderline (LMP) tumour.
  • LMP borderline
  • kits or devices for the classification of an ovarian tumour of any one disease or condition as taught herein comprising means for detecting the level of any one or more biomarkers in a sample of the patient.
  • a kit or kits of the invention can be used in clinical settings or at home.
  • the kit according to the invention can be used for monitoring the evolution or classifying an ovarian tumour in a subject or for preventive screening of subjects belonging to a high risk group for instance for the occurrence or recurrence of an ovarian tumour in said subject.
  • kits or devices comprise the following elements: a) a means for obtaining a sample from the subject b) a means or device for measuring the amount of any one or more markers as taught herein in said sample and visualizing whether the amount of the one or more markers in said sample is below or above a certain threshold level or value, indicating the occurrence or recurrence of an ovarian tumour in said subject or indicating a change in the classification of an ovarian tumour in said subject as taught herein.
  • threshold level or value or “reference value” is used interchangeably as a synonym and is as defined herein. It can also be a range of base-line (e.g. "dry weight”) values determined in an individual patient or in a group of patients with highly similar disease conditions, or it can be a risk scale on a risk stratification tool as taught herein.
  • the inventors saw that the Cystatin-C level is decreased in case of a malignant ovarian tumour, both at the protein and mRNA level. In the patients tested, the Cystatin-C level is lower than that of subjects with a benign ovarian tumour. Furthermore, the inventors saw that the Cystatin-C level is increased in case of a benign ovarian tumour, both at the protein and mRNA level. In the patients tested, the Cystatin-C level is higher than that of subjects with a malignant ovarian tumour.
  • the threshold value indicated in the present invention is therefore more to be seen as a value in a reference, i.e. a subject with a malignant or benign ovarian tumour or a subclass thereof, taken at about the same stage of gestation and not so much as the value of the subject.
  • modulate generally denotes a qualitative or quantitative alteration, change or variation specifically encompassing both increase (e.g. activation) and decrease (e.g., inhibition) of that which is being modulated.
  • increase e.g. activation
  • decrease e.g., inhibition
  • the term encompasses any extent of such modulation.
  • modulation may encompass an increase in the value of said variable by at least about 10%, e.g., by at least about 20%, preferably by at least about 30%, e.g., by at least about 40%, more preferably by at least about 50%, e.g., by at least about 75%, even more preferably by at least about 100%, e.g., by at least about 150%, 200%, 250%, 300%, 400% or by at least about 500%, compared to a reference situation without said modulation; or modulation may encompass a decrease or reduction in the value of said variable by at least about 10%, e.g., by at least about 20%, by at least about 30%, e.g., by at least about 40%, by at least about 50%, e.g., by at least about 60%, by at least about 70%, e.g., by at least about 80%, by at least about 90%, e.g., by at least about 95%, such as by
  • modulation of the activity and/or level of intended target(s) may be specific or selective, i.e., the activity and/or level of intended target(s) may be modulated without substantially altering the activity and/or level of random, unrelated (unintended, undesired) targets.
  • intended target(s) e.g., Cystatin-C gene or protein
  • modulation of the activity and/or level of intended target(s) may be specific or selective, i.e., the activity and/or level of intended target(s) may be modulated without substantially altering the activity and/or level of random, unrelated (unintended, undesired) targets.
  • Reference to the "activity" of a target such as Cystatin-C protein may generally encompass any one or more aspects of the biological activity of the target, such as without limitation any one or more aspects of its biochemical activity, enzymatic activity, signalling activity and/or structural activity, e.g., within a cell, tissue, organ or an organism.
  • the reference to the "level" of a target such Cystatin-C gene or protein may preferably encompass the quantity and/or the availability (e.g., availability for performing its biological activity) of the target, e.g., within a cell, tissue, organ or an organism.
  • the level of a target may be modulated by modulating the target's expression and/or modulating the expressed target. Modulation of the target's expression may be achieved or observed, e.g., at the level of heterogeneous nuclear RNA (hnRNA), precursor mRNA (pre-mRNA), mRNA or cDNA encoding the target.
  • hnRNA heterogeneous nuclear RNA
  • pre-mRNA precursor mRNA
  • mRNA or cDNA encoding the target e.g., at the level of heterogeneous nuclear RNA (hnRNA), precursor mRNA (pre-mRNA), mRNA or cDNA encoding the target.
  • decreasing the expression of a target may be achieved by methods known in the art, such as, e.g., by transfecting (e.g., by electroporation, lipofection, etc.) or transducing (e.g., using a viral vector) a cell, tissue, organ or organism with an antisense agent, such as, e.g., antisense DNA or RNA oligonucleotide, a construct encoding the antisense agent, or an RNA interference agent, such as siRNA or shRNA, or a ribozyme or vectors encoding such, etc.
  • an antisense agent such as, e.g., antisense DNA or RNA oligonucleotide, a construct encoding the antisense agent, or an RNA interference agent, such as siRNA or shRNA, or a ribozyme or vectors encoding such, etc.
  • increasing the expression of a target may be achieved by methods known in the art, such as, e.g., by transfecting (e.g., by electroporation, lipofection, etc.) or transducing (e.g., using a viral vector) a cell, tissue, organ or organism with a recombinant nucleic acid which encodes said target under the control of regulatory sequences effecting suitable expression level in said cell, tissue, organ or organism.
  • the level of the target may be modulated via alteration of the formation of the target (such as, e.g., folding, or interactions leading to formation of a complex), and/or the stability (e.g., the propensity of complex constituents to associate to a complex or disassociate from a complex), degradation or cellular localisation, etc. of the target.
  • said modulation leads to an increase in Cystatin-C activity, either by activating its function it at the protein level or by upregulating transcription and translation of the coding sequence of Cystatin-C into its protein, i.e. at the mRNA or gene level. Since it is clear that the Cystatin-C level is decreased in subjects with a malignant ovarian tumour as defined herein, increasing the activity of Cystatin-C intends to improve the condition of the subject.
  • antisense generally refers to a molecule designed to interfere with gene expression and capable of specifically binding to an intended target nucleic acid sequence.
  • Antisense agents typically encompass an oligonucleotide or oligonucleotide analogue capable of specifically hybridising to the target sequence, and may typically comprise, consist essentially of or consist of a nucleic acid sequence that is complementary or substantially complementary to a sequence within genomic DNA, hnRNA, mRNA or cDNA, preferably mRNA or cDNA corresponding to the target nucleic acid.
  • Antisense agents suitable herein may typically be capable of hybridising to their respective target at high stringency conditions, and may hybridise specifically to the target under physiological conditions.
  • ribozyme generally refers to a nucleic acid molecule, preferably an oligonucleotide or oligonucleotide analogue, capable of catalytically cleaving a polynucleotide.
  • a "ribozyme” may be capable of cleaving mRNA of a given target protein, thereby reducing translation thereof.
  • Exemplary ribozymes contemplated herein include, without limitation, hammer head type ribozymes, ribozymes of the hairpin type, delta type ribozymes, etc. For teaching on ribozymes and design thereof, see, e.g., US 5,354,855, US 5,591 ,610, Pierce et al.
  • RNA interference or "RNAi” technology is routine in the art, and suitable RNAi agents intended herein may include inter alia short interfering nucleic acids (siNA), short interfering RNA (siRNA), double-stranded RNA (dsRNA), micro-RNA (miRNA), and short hairpin RNA (shRNA) molecules as known in the art.
  • siNA short interfering nucleic acids
  • siRNA short interfering RNA
  • dsRNA double-stranded RNA
  • miRNA micro-RNA
  • shRNA short hairpin RNA
  • carrier or “excipient” includes any and all solvents, diluents, buffers (such as, e.g., neutral buffered saline or phosphate buffered saline), solubilisers, colloids, dispersion media, vehicles, fillers, chelating agents (such as, e.g., EDTA or glutathione), amino acids (such as, e.g., glycine), proteins, disintegrants, binders, lubricants, wetting agents, emulsifiers, sweeteners, colorants, flavourings, aromatisers, thickeners, agents for achieving a depot effect, coatings, antifungal agents, preservatives, antioxidants, tonicity controlling agents, absorption delaying agents, and the like.
  • compositions for pharmaceutical active substances.
  • present active substances agents
  • therapeutic agents may be used alone or in combination with any therapies known in the art for the disease and conditions as taught herein ("combination therapy").
  • Combination therapies as contemplated herein may comprise the administration of at least one active substance of the present invention and at least one other pharmaceutically or biologically active ingredient.
  • Said present active substance(s) and said pharmaceutically or biologically active ingredient(s) may be administered in either the same or different pharmaceutical formulation(s), simultaneously or sequentially in any order.
  • the dosage or amount of the present active substances (agents) used, optionally in combination with one or more other active compound to be administered, depends on the individual case and is, as is customary, to be adapted to the individual circumstances to achieve an optimum effect. Thus, it depends on the nature and the severity of the disorder to be treated, and also on the age, body weight, general health, diet, mode and time of administration, and individual responsiveness of the human or animal to be treated, on the route of administration, efficacy, metabolic stability and duration of action of the compounds used, on whether the therapy is acute or chronic or prophylactic, or on whether other active compounds are administered in addition to the agent(s) of the invention.
  • a phrase such as "a subject in need of treatment” includes subjects that would benefit from treatment of an ovarian tumour as taught herein. Such subjects may include, without limitation, those that have been diagnosed with an ovarian tumour, those that have undergone surgery of an ovarian tumour or those belonging to a high risk group of developing an ovarian tumour.
  • treat or “treatment” both encompass the therapeutic treatment of an already developed disease or condition, as well as prophylactic or preventative measures, wherein the aim is to prevent or lessen the chances of incidence of an undesired affliction, such as to prevent progression of an ovarian tumour as taught herein.
  • Beneficial or desired clinical results may include, without limitation, alleviation of one or more symptoms or one or more biological markers, diminishment of extent of disease, stabilised (i.e., not worsening) state of disease, delay or slowing of disease progression, amelioration or palliation of the disease state, and the like.
  • Treatment can also mean prolonging survival as compared to expected survival if not receiving treatment.
  • prophylactically effective amount refers to an amount of an active compound or pharmaceutical agent that inhibits or delays in a subject the onset of a disorder as being sought by a researcher, veterinarian, medical doctor or other clinician.
  • therapeutically effective amount refers to an amount of active compound or pharmaceutical agent that elicits the biological or medicinal response in a subject that is being sought by a researcher, veterinarian, medical doctor or other clinician, which may include inter alia alleviation of the symptoms of the disease or condition being treated. Methods are known in the art for determining therapeutically and prophylactically effective doses for the present compounds. The above aspects and embodiments are further supported by the following non-limiting examples.
  • Example 1 Samples: cohort overview The cohort used in this experiment was a prospective multicenter study. All patients were diagnosed with a pelvic mass of suspected ovarian origin and were scheduled for surgical intervention. All patients underwent imaging by pelvic ultrasound to document the presence of an ovarian mass. All patients underwent surgical removal of the ovarian mass and these were examined histologically to make a final histopathological diagnosis, wherein patients were classified as having benign or malignant tumours.
  • Tablel Distribution of patient characteristics for patients with a benign or malignant pelvic mass
  • Table 2 histological type of benign disease and stage and grade for malignant disease
  • EOC epithelial ovarian carcinoma
  • serum and plasma was prepared.
  • CA125 levels were measured either using the Roche assay (further referred to as central lab measurement) or the CanAg CA125 EIA assay (Fujirebio Diagnostics, Goteborg, Sweden).
  • Serum HE4 concentrations were measured using the HE4 EIA assay (Fujirebio Diagnostics).
  • Cystatin C levels were determined using MASSterclass ® as outlined in example 2.
  • MASSterclass® assays use targeted tandem mass spectrometry with stable isotope dilution as an end-stage peptide quantitation system (also called Multiple Reaction Monitoring (MRM) and Single Reaction Monitoring (SRM).
  • MRM Multiple Reaction Monitoring
  • SRM Single Reaction Monitoring
  • the targeted peptide is specific ⁇ i.e., proteotypic) for the specific protein of interest, i.e., the amount of peptide measured is directly related to the amount of protein in the original sample.
  • peptide fractionation precedes the end-stage quantitation step.
  • a suitable MASSTERCLASS® assay may include the following steps:
  • isotopically labelled peptides has the same amino acid sequence as the proteotypic peptides of interest, typically with one isotopically labelled amino acid built in to generate a mass difference.
  • the labelled peptide has identical chemical and chromatographic behaviour as the endogenous peptide, except during the end-stage quantitation step which is based on molecular mass.
  • the proteins in the depleted serum/plasma sample are digested into peptides using trypsin. This enzyme cleaves proteins C-terminally from lysine and arginine, except when a proline is present C-terminally of the lysine or arginine.
  • proteins are denatured by boiling, which renders the protein molecule more accessible for the trypsin activity during the 16h incubation at 37°C.
  • - Peptide-based fractionation The charged peptide molecules are separated based on their specific isoelectric property. As there is no pi difference between the endogenous peptide and the isotopically labelled variant, they co-elute. Only those fractions containing the monitored peptides, or pools thereof, are selected and proceed to the next level of fractionation.
  • the LC column is connected to an electrospray needle connected to the source head of the mass spectrometer.
  • the peptide that is monitored is specifically selected to pass the first quadrupole (Q1 ), based on its mass to charge ratio (m/z).
  • the selected peptide is then fragmented in a second quadrupole (Q2) which is used as a collision cell.
  • the resulting fragments then enter the third quadrupole (Q3).
  • a specific peptide fragment or specific peptide fragments are selected for detection.
  • transition The combination of the m/z of the monitored peptide and the m/z of the monitored fragment of this peptide is called a transition. This process can be performed for multiple transitions during one experiment. Both the endogenous peptide (analyte) and its corresponding isotopically labelled synthetic peptide (internal standard) elute at the same retention time, and are measured in the same LC-MS/MS experiment.
  • the MASSterclass® readout is defined by the ratio between the area under the peak specific for the analyte and the area under the peak specific for the synthetic isotopically labelled analogue (internal standard). MASSterclass® readouts are directly related to the original concentration of the protein in the sample. MASSterclass® readouts can therefore be compared between different samples and groups of samples.
  • - 25 ⁇ _ of plasma is subjected to a depletion of human albumin and IgG (ProteoPrep spin columns; Sigma Aldrich) according to the manufacturer's protocol, except that 20mM NH4HCO 3 was used as the binding/equilibration buffer.
  • the depleted sample (225 ⁇ _) is denatured for 15min at 95°C and immediately cooled on ice - 2 pmol of each isotopically labelled peptide (custom made 'Heavy AQUA' peptide; Thermo Scientific) is spiked in the sample
  • method contains the transitions for the analyte as well as for the synthetic, labelled peptide.
  • Each of the transitions of interest was measured for a period starting 2 to 3 minutes before and ending 2 to 3 minutes after the determined retention time of the peptide of interest, making sure that each peak had at least 10 datapoints.
  • the MASSterclass® readout was defined by the ratio of the analyte peak area and the internal standard peak area
  • the measured ratios are differential quantitations of peptides.
  • a ratio is the normalised concentration of a peptide.
  • concentration of a peptide is proportional to the ratio measured in the mass spectrometer.
  • Example 3 Cystatin C adds value to existing ovarian markers to discriminate malignant from benign disease
  • a coefficient for each variable included in the model as well as a model constant was calculated.
  • a predicted probability was calculated for each patient using each of the logistic regression models, the resulting predicted probability values ranging from 0% to 100% for each model. For all statistical comparisons a level of p ⁇ 0.05 was accepted statistically significant.
  • Table 3 Significance of added value of Cystatin C to CA125 and HE4 markers to discriminate benign ovarian tumours from malignant ovarian tumours
  • Table 5 Significance of added value of eGfr to CA125, HE4 and Cystatin C markers to discriminate benign ovarian tumours from malignant ovarian tumours
  • Table 5 summarizes the impact of adding eGfr to the equation illustrating that normalizing cystatin C for its filtration further improves its diagnostic potential.
  • Said analysis is used to compare the performance of the risk stratification tool using CA125 and HE4 (known as the ROMA test) and the clinically used Risk of Malignancy Index (RMI), with the newly developed risk stratification tool of the present invention comprising the measurement of Cystatin C, optionally compensated by the measured eGfr value, in combination with HE4, CA125, or in combination with both HE4 and CA125.
  • CA125 and HE4 known as the ROMA test
  • RMI Risk of Malignancy Index
  • the analysis will test whether adding Ultrasound Data to the stratification tool of the invention would improve its specificity and/or sensitivity.

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Abstract

L'invention concerne la Cystatine C ou des fragments de celle-ci comme nouveau biomarqueur pour la classification d'une tumeur ovarienne chez un sujet ; des procédés de classification d'une tumeur ovarienne chez un sujet sur la base de la mesure dudit biomarqueur ; et des trousses et dispositifs pour la mesure dudit biomarqueur et/ou la mise en œuvre desdits procédés.
PCT/EP2012/065610 2011-08-12 2012-08-09 Nouveau biomarqueur pour la classification de tumeurs ovariennes Ceased WO2013023994A1 (fr)

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2017005974A1 (fr) * 2015-07-03 2017-01-12 Turun Yliopisto Diagnostics de maladies gynécologiques, en particulier du cancer épithélial de l'ovaire
WO2017219093A1 (fr) * 2016-06-24 2017-12-28 Macquarie University Procédés de criblage
CN108152430A (zh) * 2017-12-21 2018-06-12 上海中科新生命生物科技有限公司 基于prm检测的卵巢癌标志物检测试剂盒及检测方法
WO2020193403A1 (fr) * 2019-03-22 2020-10-01 Katholieke Universiteit Leuven Méthodes de diagnostic du cancer de l'ovaire
CN115629214A (zh) * 2022-12-21 2023-01-20 北京大学第三医院（北京大学第三临床医学院） 一种用于卵巢癌早期诊断的生物标志物及其应用
US20230176073A1 (en) * 2018-03-13 2023-06-08 Government Of The United States, As Represented By The Secretary Of The Army Assays and methods for targeted treatment of hydrosalpinx

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Cited By (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2017005974A1 (fr) * 2015-07-03 2017-01-12 Turun Yliopisto Diagnostics de maladies gynécologiques, en particulier du cancer épithélial de l'ovaire
US10684285B2 (en) 2015-07-03 2020-06-16 Kaivogen Oy Diagnostics of gyneacological diseases, especially epithelial ovarian cancer
WO2017219093A1 (fr) * 2016-06-24 2017-12-28 Macquarie University Procédés de criblage
CN108152430A (zh) * 2017-12-21 2018-06-12 上海中科新生命生物科技有限公司 基于prm检测的卵巢癌标志物检测试剂盒及检测方法
US20230176073A1 (en) * 2018-03-13 2023-06-08 Government Of The United States, As Represented By The Secretary Of The Army Assays and methods for targeted treatment of hydrosalpinx
US12287341B2 (en) 2018-03-13 2025-04-29 Government Of The United States, As Represented By The Secretary Of The Army Assays and methods for targeted treatment of hydrosalpinx
WO2020193403A1 (fr) * 2019-03-22 2020-10-01 Katholieke Universiteit Leuven Méthodes de diagnostic du cancer de l'ovaire
CN115629214A (zh) * 2022-12-21 2023-01-20 北京大学第三医院（北京大学第三临床医学院） 一种用于卵巢癌早期诊断的生物标志物及其应用

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