A method for early detection of ectopic endometrium

Abstract

The present disclosure provides a method that can be used in the early detection of ectopic endometrium (adenomyosis and / or endometriosis) comprising using contrast-enhanced ultrasound (CEUS) images of uterine vasculature of a subject for qualitative and quantitative analysis of enhancement patterns.

Description

Title: A METHOD FOR EARLY DETECTION OF ECTOPIC ENDOMETRIUMTECHNICAL FIELD[1] The present disclosure relates generally to the field of medicine and more specifically to gynecology. More particularly, the present disclosure provides a method that can be used in the early detection of ectopic endometrium which characterizes disorders such as but not limited to adenomyosis and endometriosis.BACKGROUND[2] Heavy and painful menstruations, as well as subfertility, profoundly affect the lives of many people. Adenomyosis or endometriosis that are characterized by ectopic endometrium may be a root cause of these symptoms.[3] Both endometriosis and adenomyosis are benign disorders that are characterized by ectopic endometrium (localization at another place then the myometrium layer). Adenomyosis is a benign disorder characterized by invasion of endometrial tissue from the endometrial layer (inner lining uterus) into the uterine muscle wall. It is a common disorder, yet it is unknown to both patients as well as many health care workers. This is despite the 34% prevalence of adenomyosis in 18 to 30 year old women who attended an outpatient clinic for contraception counselling, gynaecological symptoms or fertility. Endometriosis is characterized by endometrium localized outside the uterus, this can be at different locations in the pelvis (such as bowel, bladder, between the uterus and rectum, in the ovary or at the peritoneum) and rarely, even outside the pelvis (abdominal wall, diaphragm, retroperitoneal spaces).[4] Due to the diffuse character of ectopic endometrium and inadequate imaging techniques, diagnosis of disorders such as adenomyosis and endometriosis is frequently missed, and women suffer from unexplained heavy bleeding complaints and / or infertility for years. The average time from symptoms to diagnosis for endometriosis is eight years, according to a parliamentary report from the UK in 2020. While for adenomyosis these numbers have not even been estimated, but they are expected to be at least as long.[5] Sometimes, adenomyosis causes no signs or symptoms or only mild discomfort. However, adenomyosis can cause: heavy or prolonged menstrual bleeding, severe cramping or sharp, knifelike pelvic pain during menstruation (dysmenorrhea), chronic pelvic pain, painful intercourse (dyspareunia). In some cases, the uterus is enlarged. Anenlarged uterus may result in tenderness or pressure in the lower abdomen. Known symptoms of adenomyosis include painful menstrual cramps (dysmenorrhea), heavy menstrual bleeding (menorrhagia), abnormal menstruation, pelvic pain, painful intercourse (dyspareunia), subfertility (reduced fertility), infertility, increased miscarriage rate and an enlarged uterus and is associated with preterm birth. Symptoms associated with endometriosis are the same but dependent on its localization may also include bladder or defecations problems or pain during or after miction or defecation. The symptoms have a considerable impact on quality of life, including work-life, fertility (in view of associated risks of infertility and miscarriages) and healthcare and societal costs.[6] The main reason for the unfamiliarity with adenomyosis and endometriosis is that uniform diagnostic tools are lacking. With the advancements of gynecological sonography, specific features of adenomyosis can be recognized on conventional ultrasound and have enabled specialized gynecologists in academic centers to diagnose adenomyosis without surgically removing the uterus and endometriosis without performing a laparoscopy. Yet, there is a strong need for advanced imaging techniques clearly demonstrating the presence of ectopic endometrium. While methods for diagnosis of Deep Endometriosis (DE) and other conditions such as cancer stemming from localized fibrosis (also associated with severe forms of endometriosis like DE) has been previously discussed in the art - see for instance Zheng et al. and Kuenen et. al.2, there still is a need to devise methods that facilitate detection of these conditions at an early stage.[7] Given the impact the disease has on the quality of life, fertility, healthcare and societal costs as described above, it is pertinent to detect the disease at an early stage to ensure that the quality of life and societal participation of patients is not compromised. The present disclosure addresses the said need in the art.BRIEF DESCRIPTION OF THE FIGURES[8] In order that the disclosure may be readily understood and put into practical effect, reference will now be made to exemplary embodiments as illustrated with reference to the accompanying figures. The figures together with detailed description below, are incorporated in and form part of the specification, and serve to further illustrate the embodiments and explain various principles and advantages, where:[9] FIGURE 1 depicts B-mode (left) and CEUS (right) of a sagittal section of the uterus (solid and dashed delineation) at peak intensity for the healthy control (A) and three adenomyosis patients (B, C, D).

[0010] FIGURE 2 depicts B-mode ultrasound and corresponding CUDI spatial similarity parametric maps of a normal uterus. On the B-mode, the uterine corpus and there within the endometrium are delineated.

[0011] FIGURE 3 depicts B-mode ultrasound and corresponding CUDI spatial similarity parametric maps of a very early stage adenomyotic uterine corpus with enhanced angiogenesis in the inner myometrium. On the B-mode, the uterine corpus and there within the endometrium are delineated.

[0012] FIGURE 4 depicts B-mode ultrasound and corresponding CUDI spatial similarity parametric maps of a moderate stage adenomyotic uterine corpus with enhanced angiogenesis in the inner and middle myometrium. On the B-mode, the uterine corpus and there within the endometrium are delineated.

[0013] FIGURE 5 depicts B- mode ultrasound and corresponding CUDI spatial similarity parametric maps of an advanced adenomyotic uterine corpus with decreased angiogenesis of all myometrium layers. On the B-mode, the uterine corpus and there within the endometrium are delineated.

[0014] FIGURE 6 depicts CUDI (CEUS) early (left) versus advanced (right) stage angiogenesis: a) normal homogeneous, b) heterogeneous, hyperenhanced inner and middle layer, c) all layers heterogeneous, hyperenhanced and d) homogeneous: hypoenhanced (decreased almost absent vascularity).SUMMARY OF THE INVENTION

[0015] Addressing the aforesaid need in the art, the present disclosure provides a method for early detection of ectopic endometrium, comprising: using contrast-enhanced ultrasound (CEUS) images of uterine vasculature of a subject for quantitative and qualitative analysis of enhancement patterns.

[0016] More particularly, provided herein is a method for early detection of ectopic endometrium, characterized by quantitative and qualitative analysis of enhancement patterns on CEUS images of uterine vasculature of a subject; wherein the detection is during the angiogenic phase, prior to onset of fibrosis.

[0017] The present disclosure is based on the finding that CEUS may be improvised to identify differences in vasculature between healthy and angiogenic uterine tissue. In some embodiments, angiogenic uterine tissue is adenomyotic uterine tissue or endometriosis tissue. Thus, in some embodiments, the early detection of ectopic endometrium is indicative of adenomyosis or endometriosis, preferably early stages thereof.

[0018] In some embodiments, the quantitative analysis comprises one or more of: a) analysis of pixel-based spatial similarity using contrast-ultrasound dispersion imaging (CUDI); wherein the said analysis comprises determining one or more spatial similarity metrics selected from spectral coherence (p), correlation (r), and mutual information (I); and b) time-intensity analysis of ultrasound contrast agent (UCA) wash-in and wash-out.

[0019] In some embodiments, the spectral coherence (p), the correlation (r), and the mutual information (I) are inversely proportional to dispersion; and wherein low dispersion is indicative of angiogenesis associated with early pathophysiology of ectopic endometrium, and preferably adenomyosis or endometriosis.

[0020] In some embodiments, the qualitative analysis comprises visual assessment of one or more of: a. differences in vasculature between healthy and ectopic endometrial tissue; b. differences in vasculature between different uterine layers; c. signal intensity in a specific region of interest (ROI); and d. homogeneity of myometrium enhancement; and e. enhancement of pelvic organs.

[0021] In some embodiments, the healthy uterine tissue is of the same or a different subject; and the myometrium of a subject having ectopic endometrium shows heterogeneous enhancement.

[0022] In some embodiments, the signal intensity is subjected to CEUS time-intensity curve fitting.

[0023] Further provided herein is a method of differentiating between different stages, preferably early and later stages of adenomyosis or endometriosis comprising using CEUS images of uterine vascular structures of a subject and comparing variation in CEUS enhancement patterns between healthy and adenomytic or endometriosis tissues. In a preferred embodiment, the early stage of adenomyosis or endometriosis is at an angiogenic phase, prior to onset of fibrosis.

[0024] In some embodiments, early stage adenomyosis or endometriosis is characterized by ectopic endometrium with enhanced angiogenesis compared to normal myometrium, without fibrosis.

[0025] In some embodiments, the CEUS images as referred to above are obtained from CEUS optimized for uterine vasculature. Preferably, the CEUS images referred to above are obtained from imaging performed while maintaining one or more parameters selected from:- gain of about 25 dB to about 35dB,- dynamic range of about 40 dB to about 50 dB, and- mechanical index of about 0.01 to about 0.15.

[0026] Further provided herein is a non-steroidal angiogenesis inhibitor for use in the treatment or prevention of early stage adenomyosis or early-stage endometriosis in a patient diagnosed by the method as described above.

[0027] In some embodiments, the non-steroidal angiogenesis inhibitor is administered intra-uterine.

[0028] In some embodiments, the angiogenesis inhibitor is a small molecule or an antigen binding molecule such as an antibody or antigen binding fragment thereof.DETAILED DESCRIPTION

[0029] It is an object of the present disclosure to accurately characterize and identify ectopic endometrium.

[0030] Endometriosis and adenomyosis are non-limiting examples of uterine disorders that are closely linked and characterized by ectopic endometrium. It is therefore a further object of the invention to detect conditions such as adenomyosis and endometriosis that are characterized by ectopic endometrium.

[0031] Accordingly, one of the objects of the present disclosure is to provide a method that allows discrimination between adenomyotic and healthy tissue.

[0032] A further object of the present disclosure is to provide a method that allows discrimination between endometriosis and healthy tissue.

[0033] To survive, ectopic endometrium must connect to the vascular system by angiogenesis. Accordingly, it is also an object of the present disclosure to provide amethod that detects enhancement in angiogenesis that is indicative of ectopic endometrium or conditions such as adenomyosis and endometriosis stemming from the same.

[0034] It is also an object of the present disclosure to provide an improvement over existing detection techniques to allow early detection of enhanced angiogenesis and identification of adenomyotic tissue or endometriosis tissue.

[0035] A further objective of the present disclosure is to identify ectopic endometrium outside the uterus (i.e. endometriosis) based on increased or decreased angiogenesis compared to normal surrounding tissue.

[0036] Another object of the present disclosure is to provide an upgrade to the currently available scanning techniques such as contrast-enhanced ultrasound (CEUS) to ensure that they are specific enough for visualizing and identifying ectopic uterus.

[0037] It is a further object of the present disclosure to provide an upgrade to the currently available scanning techniques such as contrast-enhanced ultrasound (CEUS) to ensure that they are specific enough for visualizing, identifying and distinguishing between adenomyotic tissue and endometriosis tissue.

[0038] A further objective of the present disclosure is to determine optimal machine settings and feasibility of uterine CEUS to identify ectopic endometrium and associated disorders such as adenomyosis and endometriosis.

[0039] Another objective of the present disclosure is to visualize and describe the adenomyotic uterus or endometriotic lesions both qualitatively and quantitatively using CEUS images.

[0040] A further object of the present disclosure is to provide a CEUS-based prediction model, based on such qualitative and quantitative analysis for the early detection of adenomyosis and endometriosis and to predict the responsiveness to therapies such as angiogenesis inhibiting therapies.

[0041] The present disclosure thus provides a straightforward CEUS-based method for use in the analysis of the uterine vascular architecture to allow detection and characterization of ectopic endometrium and associated disorders such as adenomyosis or endometriosis.

[0042] However, before describing the subject matter of the present disclosure in further detail, the below paragraphs set out definitions for some terms used throughout the present disclosure for the purposes of clarity and convenience. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understoodby one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, the preferred methods and materials are now described.Definitions

[0043] As used herein the term “ectopic endometrium” refers to endometrium outside the normal endometrial layer. If ectopic endometrium is located in the uterus it is called adenomyosis, when it is located outside the uterus it is called endometriosis.

[0044] As used herein, the term “adenomyosis” refers to a benign condition where endometrium tissue is present in the myometrium layer of the uterus. Adenomyosis occurs when the endometrial tissue that normally lines the uterine cavity grows into the muscular layers of the uterus. The displaced tissue continues to act on hormonal changes during each menstrual cycle, including thickening, breaking down and bleeding. As a result, ectopic endometrium may enlarge the uterus and may induce painful and heavy periods can occur. It also induces inflammatory actions when it is at a different location and may induce abnormal contractions resulting in lower implantation rates and higher miscarriage rates and possibly also higher rate of preterm birth. Ectopic endometrium outside the uterus may induce bleeding, development of adhesions or fibrosis in or between other organs (bladder / bowel / skin) and induce heavy pain, dependent on its localization.

[0045] As used herein, the term “endometriosis” refers to a condition wherein tissue similar to the lining of the uterus grows outside of the uterus. With endometriosis, the tissue can be found on the ovaries, fallopian tubes or the intestines. Common symptoms of endometriosis include painful periods and heavy menstrual bleeding.

[0046] Reference to “early” in the context of detection of ectopic endometrium, adenomyosis or endometriosis, implies detection during the angiogenic phase when no fibrosis is present. Reference to “early” in the context of detection of adenomyosis or endometriosis implies detection of the said conditions at early stages of the disease, during the angiogenic phase i.e. when there is enhanced angiogenesis, and when no fibrosis is present.

[0047] As used herein, the term “contrast-enhanced ultrasound” or “CEUS” refers to the application of ultrasound contrast medium to traditional medical sonography. Said technique helps better visualize organs and blood vessels within the abdomen and pelvis.

[0048] The term “contrast-ultrasound dispersion imaging" or “CUDI” as used in the present disclosure refers to a technique to assist in the interpretation, particularly, quantitative analysis of CEUS results. It is based on the fact that angiogenic vascular structures are small in diameter, compressible, highly tortuous and lack normal endothelial lining, leading to less efficient dispersion of blood / contrast. In the present disclosure, CUDI has been used for pixel-based spatial similarity analysis of CEUS image(s), to determine spatial similarity metrics selected from spectral coherence (p), correlation (r), and mutual information (I).

[0049] As used herein, the term “enhancement pattern” refers to a pattern of increase in signal intensity in a specific area on an ultrasound image following the intake of contrast material. Enhancement can help identify areas of abnormal tissue that have increased blood flow and uptake of the contrast material.

[0050] The term “angiogenesis” refers to the physiological process through which new blood vessels form from pre-existing vessels. Angiogenesis continues the growth of the vasculature by processes of sprouting and splitting. It is a sophisticated process, regulated by the balance between endogenous, pro-angiogenic (or stimulatory) and anti-angiogenic (or inhibitory) factors.

[0051] The term “fibrosis” refers to a pathological response to tissue damage leading to excessive formation of connective tissue to replace the damaged tissue, that in turn results in thickening or scarring of the tissue.

[0052] As used herein, the term "dispersion", “dispersed” or obvious variants thereof relates to spreading of the contrast agents through the vascular network.

[0053] The term “vascularization”, as used herein, refers to the process of growing blood vessels into a tissue to improve oxygen and nutrient supply. The term “uterine vasculature” refers to the vascular system of the uterus, wherein analysis of the same forms a key feature of the present disclosure. The term “uterine vasculature” also encompasses uterine microvascularization.

[0054] The term “uterine microvascularization” or “uterine vasculature” in the context of the present disclosure refers to arterial and venous patterns in human uteri. In the context of the present disclosure, said term has been used while describing uterine microanatomical vascular visualization that has been relied upon in the method(s) as provided herein.

[0055] The term “equivalent setting on an Ultrasound system” as used herein refers to a setting in an Ultrasound device suitable for CEUS imaging which provides a comparable result as the setting in a Samsung’s HERA W10. A skilled person / team may calibrate the settings to obtain the same results as described herein to obtain the equivalent setting referred to. The said settings may be employed for imaging irrespective of the specific kind of probe used, examples of which include abdominal and endo vaginal probes.

[0056] The term “treating” as used herein refers to a reduction of the severity of a benign uterine disorder, and / or a reduction of growth or induction of regression of ectopic endometrial tissue (endometrial tissue growing into the muscular wall of the uterus or in organs outside the uterus in case of endometriosis) and / or reduction of one or more of the symptoms associated with a benign uterine disorder and / or restoration of fertility. Preferably, said treatments result in restoration or improvement of the health of a subject or individual. Preferably, the treated subject or individual demonstrates fewer disease symptoms or experiences disease symptoms for a shorter time. Preferably, said treatments result in restoring fertility, increasing fertility, increasing pregnancy rates, and / or reducing risk of miscarriage or preterm birth.

[0057] The term “preventing” as used herein refers to the prevention of development of a benign uterine disorder and / or prevention of growth of the endometrial tissue into the muscular wall of the uterus and / or prevention of one or more of the symptoms associated with a uterine being disorder and / or prevention against loss of fertility. As recognized by a skilled person, prevention does not mean that none of the treated females will ever develop said disorder. Rather, prevention refers to a decreased likelihood that a female with develop said disorder or symptoms as compared to a non-treated female.

[0058] As used herein, the term "subject" is a vertebrate, such as a mammal, such as a human. Mammals include, but are not limited to, humans, livestock, athletic animals, pets and the like.

[0059] As used herein, the term “comprising” when placed before the recitation of steps in a method means that the method encompasses one or more steps that are additional to those expressly recited, and that the additional one or more steps may be performed before, between, and / or after the recited steps. For example, a method comprising steps a, b, and c encompasses a method of steps a, b, x, and c, a method of steps a, b, c, and x, as well as a method of steps x, a, b, and c. Furthermore, the term “comprising” when placed before the recitation of steps in a method does not (although it may) require sequentialperformance of the listed steps, unless the content clearly dictates otherwise. For example, a method comprising steps a, b, and c encompasses, for example, a method of performing steps in the order of steps a, c, and b, the order of steps c, b, and a, and the order of steps c, a, and b, etc.

[0060] With respect to the use of substantially any plural and / or singular terms herein, those having skill in the art can translate from the plural to the singular and / or from the singular to the plural as is appropriate to the context and / or application. The various singular / plural permutations may be expressly set forth herein for sake of clarity. The suffix “(s)” at the end of any term in the present disclosure envisages in scope both the singular and plural forms of said term.

[0061] As used in this specification and the appended claims, the singular forms “a,” “an” and “the” includes both singular and plural references unless the content clearly dictates otherwise. For example, the term “inserted at a position” as used herein in reference to a polypeptide sequence refers to insertion at one or more (such as one, two, three, etc.) amino acid positions in the polypeptide sequence. The use of the expression ‘at least’ or ‘at least one’ suggests the use of one or more elements or ingredients or quantities, as the use may be in the embodiment of the disclosure to achieve one or more of the desired objects or results. As such, the terms “a” (or “an”), “one or more”, and “at least one” can be used interchangeably herein.

[0062] Numerical ranges stated in the form ‘from x to y’ include the values mentioned and those values that lie within the range of the respective measurement accuracy as known to the skilled person. If several preferred numerical ranges are stated in this form, of course, all the ranges formed by a combination of the different end points are also included.

[0063] The terms “about” or “approximately” as used herein when referring to a measurable value such as a parameter, an amount, a temporal duration, and the like, are meant to encompass variations of and from the specified value, such as variations of + / - 10% or less, + / -5% or less, + / -1% or less, and + / -0.1% or less of and from the specified value, insofar such variations are appropriate to perform in the disclosed invention. It is to be understood that the value to which the modifier “about” or “approximately” refers is itself also specifically, and preferably, disclosed.

[0064] As used herein, the terms “include” (any form of “include”, such as “include”), “have” (and “have”), “comprise” etc. any form of “having”, “including” (and any form of “including” such as “including”), “containing”, “comprising” or “comprises” are inclusiveand will be understood to imply the inclusion of a stated element, integer or step, or group of elements, integers or steps, but not the exclusion of any other element, integer or step, or group of elements, integers or steps

[0065] As regards the embodiments characterized in this specification, it is intended that each embodiment be read independently as well as in combination with another embodiment. For example, in case of an embodiment 1 reciting 3 alternatives A, B and C, an embodiment 2 reciting 3 alternatives D, E and F and an embodiment 3 reciting 3 alternatives G, H and I, it is to be understood that the specification unambiguously discloses embodiments corresponding to combinations A, D, G; A, D, H; A, D, I; A, E, G; A, E, H; A, E, I; A, F, G; A, F, H; A, F, I; B, D, G; B, D, H; B, D, I; B, E, G; B, E, H; B, E, I; B, F, G; B, F, H; B, F, I; C, D, G; C, D, H; C, D, I; C, E, G; C, E, H; C, E, I; C, F, G; C, F, H; C, F, I, unless specifically mentioned otherwise.DisclosureMethod of early detection of adenomyosis using CEUS images

[0066] The present disclosure provides a method for the early detection of ectopic endometrium. Ectopic endometrium is characteristic of disorders such as adenomyosis and endometriosis. Said method of the present disclosure is characterized by the use of CEUS images to identify uterine angiogenesis and optionally, fibrosis or confirm absence thereof.

[0067] Since ectopic endometrium must connect to vasculature for survival, enhanced angiogenesis may be considered as an indicator for ectopic endometrium. The present disclosure is based on the finding that CEUS may be improvised to identify differences in vasculature between healthy and angiogenic uterine tissue. In some embodiments, angiogenic uterine tissue is adenomyotic uterine tissue or endometriosis tissue.

[0068] While CEUS allows qualitative analysis by obtaining blood flow parameters, such as wash-in / wash-out rate and peak intensity, these parameters alone may not be sufficient for the detection of ectopic endometrium. The present disclosure additionally proposes quantitative analysis of CEUS images by CEUS time-intensity curve fitting and pixelbased spatial similarity analysis using contrast-ultrasound dispersion imaging (CUDI).

[0069] Thus, the present disclosure provides a method for use of CEUS in the detection of ectopic endometrium, wherein besides qualitative analysis, quantitative analysis of the CEUS images can be performed by CEUS time-intensity curve fitting and pixel-basedspatial similarity by contrast-ultrasound dispersion imaging (CUDI), resulting in metrics which have a correlation to dispersion of ultrasound. It is one of the objectives of the present disclosure to provide a method that allows early detection of ectopic endometrium. Thus, in some embodiments, the present disclosure provides a method for use of CEUS in the early detection of ectopic endometrium, wherein besides qualitative analysis, quantitative analysis of the CEUS images can be performed by CEUS time-intensity curve fitting and pixel-based spatial similarity CUDI, resulting in metrics which have a correlation to dispersion of ultrasound.

[0070] Without wishing to be bound by theory, the detection of ectopic endometrium may in turn be indicative of uterine disorders such as adenomyosis or endometriosis. Thus, more particularly, provided herein is a method for use of CEUS in the detection of adenomyosis or endometriosis, wherein besides qualitative analysis, quantitative analysis of the CEUS images can be performed by CEUS time-intensity curve fitting and pixelbased spatial similarity by CUDI, resulting in metrics which have a correlation to dispersion of ultrasound. Early detection of an ectopic endometrium, in some embodiments, further enables detection of disorders such as adenomyosis and endometriosis at early stages. Thus, in some embodiments, provided herein is a method for use of CEUS in the early detection of adenomyosis or endometriosis, wherein besides qualitative analysis, quantitative analysis of the CEUS images can be performed by CEUS time-intensity curve fitting and pixel-based spatial similarity by CUDI, resulting in metrics which have a correlation to dispersion of ultrasound.

[0071] A lower dispersion as compared to corresponding healthy tissues is associated with higher vascular density, tortuosity, and irregularity of the microvasculature, potentially indicative of angiogenic processes. As mentioned earlier, ectopic endometrium must connect to the vascular system by angiogenesis for survival. Accordingly, detection of angiogenesis may be correlated with ectopic endometrium which is an underlying characteristic of uterine disorders such as adenomyosis and endometriosis. Both adenomyosis and endometriosis have been shown to be associated with increased angiogenesis. More particularly, increased expression of angiogenic markers, as well as increased microvascular density in adenomyosis and endometriosis suggest a pivotal role for angiogenesis in the early pathophysiology of the said disorders. The improvisation to CEUS imaging and analysis proposed in the present disclosure thus makes it feasible touse uterine CEUS imaging in the detection of ectopic endometrium, preferably conditions such as adenomyosis and endometriosis that are characterized by ectopic endometrium.

[0072] The method of the present disclosure particularly uses CEUS images of uterine vasculature of a subject for qualitative and quantitative analysis of enhancement patterns. Thus, the detection as described herein bases reliance on the CEUS images of the uterine vasculature. The said CEUS images are obtained by CEUS imaging, preferably optimized for uterine vasculature, wherein the said imaging, in some embodiments, precedes or accompanies the detection. In a preferred embodiment, the CEUS imaging is performed prior to the method of detection as described herein. Thus, in some embodiments, the CEUS imaging is a method separate from the method of detection as described herein and is preferably performed prior to the method of detection that is characterized by quantitative and qualitative analysis of enhancement patterns on the CEUS images.

[0073] Accordingly, provided in the present disclosure is a method for detection of uterine angiogenesis comprising: using CEUS images of uterine vasculature of a subject for qualitative and quantitative analysis of enhancement patterns.

[0074] In some embodiments, provided in the present disclosure is a method for detection of ectopic endometrium comprising: using CEUS images of uterine vasculature of a subject for qualitative and quantitative analysis of enhancement patterns.

[0075] Put differently, in some embodiments, provided herein is a method for detection of uterine angiogenesis and preferably ectopic endometrium, characterized by quantitative and qualitative analysis of enhancement patterns on CEUS images of uterine vasculature of a subject.

[0076] In some embodiments, provided in the present disclosure is a method for early detection of ectopic endometrium comprising: using CEUS images of uterine vasculature of a subject for qualitative and quantitative analysis of enhancement patterns.

[0077] In a preferred embodiment, provided herein is a method for early detection of ectopic endometrium, characterized by quantitative and qualitative analysis of enhancement patterns on CEUS images of uterine vasculature of a subject.

[0078] In some embodiments, the method for early detection of ectopic endometrium comprises: using CEUS images of angiogenic ectopic endometrium lesions of a subject for qualitative and quantitative analysis of enhancement patterns.

[0079] In some embodiments, the method for early detection of ectopic endometrium, is characterized by quantitative and qualitative analysis of enhancement patterns on CEUS images of angiogenic ectopic endometrium lesions of a subject.

[0080] In some embodiments, the ectopic endometrium is associated with adenomyosis or endometriosis. Thus, in some embodiments, provided herein is a method for early detection of ectopic endometrium, and preferably adenomyosis or endometriosis, comprising: using CEUS images of uterine vasculature of a subject for qualitative and quantitative analysis of enhancement patterns.

[0081] In some embodiments, provided herein is a method for early detection of ectopic endometrium, and preferably adenomyosis or endometriosis, characterized by quantitative and qualitative analysis of enhancement patterns on CEUS images of uterine vasculature of a subject.

[0082] In some embodiments, provided herein is a method for early detection of adenomyosis comprising: using CEUS images of uterine vasculature of a subject for qualitative and quantitative analysis of enhancement patterns.

[0083] In some embodiments, provided herein is a method for early detection of endometriosis comprising: using CEUS images of uterine vasculature of a subject for qualitative and quantitative analysis of enhancement patterns.

[0084] In some embodiments, provided herein is a method for early detection of adenomyosis characterized by quantitative and qualitative analysis of enhancement patterns on CEUS images of uterine vasculature of a subject.

[0085] In some embodiments, provided herein is a method for early detection of endometriosis characterized by quantitative and qualitative analysis of enhancement patterns on CEUS images of uterine vasculature of a subject.

[0086] In some embodiments, the adenomyosis and the endometriosis are early stage adenomyosis and endometriosis. Without wishing to be bound by theory, in some embodiments, early stage adenomyosis and endometriosis are characterized by enhanced angiogenesis and absence of fibrosis. Later stages of adenomyosis or endometriosis, on the other hand, tend to be associated with fibrosis. The method of the present disclosure, in preferred embodiments, is particularly characterized by the early detection of ectopic endometrium, during the angiogenic phase, prior to the onset of fibrosis. Thus, in line with one one of the objectives of the present disclosure, to enable early detection of ectopic endometrium, provided herein is a method for the early detection of ectopic endometrium, characterized by the detection during the angiogenic phase, prior to the onset of fibrosis, using contrast-enhanced ultrasound (CEUS) optimized for uterine vasculature. In some embodiments, the said detection is indicative of adenomyosis or endometriosis and allows early detection of the said condition(s). Accordingly, in some embodiments, the present disclosure provides a method for the early detection of adenomyosis or endometriosis by analysis of uterine vasculature, characterized by detection during the angiogenic phase, prior to the onset of fibrosis, using contrast-enhanced ultrasound (CEUS) optimized for the uterine vasculature. Put differently, in some embodiments, the present disclosure provides a method for detection of early stage adenomyosis or early stage endometriosis by analysis of uterine vasculature, characterized by detection during the angiogenic phase, prior to the onset of fibrosis, using contrast-enhanced ultrasound (CEUS) optimized for the uterine vasculature.

[0087] In some embodiments, provided herein is a method for early detection of ectopic endometrium, characterized by quantitative and qualitative analysis of enhancement patterns on CEUS images of uterine vasculature of a subject; wherein the detection is during the angiogenic phase, prior to onset of fibrosis.

[0088] The early detection of ectopic endometrium is indicative of adenomyosis or endometriosis. Thus, in some embodiments, the present disclosure provides a method for early detection of adenomyosis or endometriosis, characterized by quantitative and qualitative analysis of enhancement patterns on CEUS images of uterine vasculature of a subject; wherein the detection is during the angiogenic phase, prior to onset of fibrosis.

[0089] In some embodiments, the present disclosure provides a method for early detection of adenomyosis, characterized by quantitative and qualitative analysis of enhancementpatterns on CEUS images of uterine vasculature of a subject; wherein the detection is during the angiogenic phase, prior to onset of fibrosis.

[0090] In some embodiments, the present disclosure provides a method for early detection of endometriosis, characterized by quantitative and qualitative analysis of enhancement patterns on CEUS images of uterine vasculature of a subject; wherein the detection is during the angiogenic phase, prior to onset of fibrosis.

[0091] In some embodiments, the CEUS images are 2D-CEUS images. In some embodiments, the 2D-CEUS images are images of uterine tissue. In a preferred embodiment, the 2D-CEUS images are images of uterine vasculature. The 2D-CEUS images, thus, in another preferred embodiment, are obtained using contrast-enhanced ultrasound (CEUS) optimized for uterine vasculature. Without wishing to be bound by theory, in some embodiments, the 2D-CEUS images may be obtained using any ultrasound system known in the art for tracking of a suitable Ultrasound Contrast Agent (UCA) administered to the subject, preferably but not exclusively using an endovaginal probe. In case of inability to use an endovaginal probe, alternatively, an abdominal probe may be used. Settings may be suitably adjusted in case of an abdominal probe.

[0092] In some embodiments, the UCA is preferably an intravascular contrast agent and allows assessment of vascularity.

[0093] In some non-limiting embodiments, the imaging may be performed while employing a transvaginal probe fixture which may be used to minimize uterine motion.

[0094] In some embodiments, the CEUS images are obtained from imaging performed while maintaining system parameters optimized for uterine tissue imaging. In some embodiments, said parameters that are optimized include but are not limited to one or more of gain, dynamic range and mechanical index. These parameters may be suitably adjusted based on the instrument used for imaging, the conditions at which the imaging is performed and the state of the subject.

[0095] As mentioned in one of the earlier paragraphs, it is an object of the present disclosure to provide suitable parameters that allows application of CEUS for imaging of uterine tissue, especially to allow identification of ectopic endometrium. Accordingly, the present disclosure provides a set of parameters for obtaining the CEUS images subjected to analysis in the method of the present disclosure.

[0096] In some embodiment the CEUS image is obtained using a Samsung’s HERA W10. Preferably, parameters defined in the subsequent embodiments are for a Samsung HERAW10. For other devices, images are obtained using equivalent settings, determination of said equivalent settings being well within the purview of knowledge of a skilled artisan in view of the values provided herein.

[0097] In a non-limiting embodiment, the CEUS images are obtained from imaging performed while maintaining gain of about 25 dB to about 35 dB for a Samsung HERA W10 or an equivalent setting on a different Ultrasound system.

[0098] In some embodiments, the CEUS images are obtained from imaging performed while maintaining gain of about 25 dB to about 30 dB, about 25 dB to about 32 dB, about 30 dB to about 35 dB, about 25 dB, about 27 dB, about 29 dB, about 31 dB, about 33 dB or about 35 dB, including ranges and values therebetween, for a Samsung HERA W10 or an equivalent setting on a different Ultrasound system.

[0099] In another non-limiting embodiment, the CEUS images are obtained from imaging performed while maintaining dynamic range of about 40 dB to about 50 dB for a Samsung HERA W10 or an equivalent setting on a different Ultrasound system.

[0100] In some embodiments, the CEUS images are obtained from imaging performed while maintaining dynamic range of about 40 dB to about 45 dB, about 42 dB to about 46 dB, about 45 dB to about 50 dB, about 40 dB, about 42 dB, about 44 dB, about 46 dB, about 48 dB or about 50 dB, including ranges and values therebetween, for a Samsung HERA W10 or an equivalent setting on a different Ultrasound system.

[0101] In yet another non-limiting embodiment, the CEUS images are obtained from imaging performed while maintaining mechanical index of about 0.05 to about 0.15 for a Samsung HERA W10 or an equivalent setting on a different Ultrasound system.

[0102] In some embodiments, the CEUS images are obtained from imaging performed while maintaining mechanical index of about 0.05 to about 0.075, about 0.075 to about 0.1, about 0.1 to about 0.15, about 0.05, about 0.1 or about 0.15, including ranges and values therebetween, for a Samsung HERA W10 or an equivalent setting on a different Ultrasound system.

[0103] In some embodiments, the CEUS images are obtained from imaging performed while maintaining one or more parameters selected from: a. gain of about 25 dB to about 35 dB for a Samsung HERA W10 or an equivalent setting on a different Ultrasound system,b. dynamic range of about 40 dB to about 50 dB for a Samsung HERA W10 or an equivalent setting on a different Ultrasound system, and c. mechanical index of about 0.05 to about 0.15 for a Samsung HERA W10 or an equivalent setting on a different Ultrasound system.

[0104] In some embodiments, the CEUS images are obtained from imaging performed while maintaining one or more parameters selected from: a. gain of about 25 dB to about 30 dB, about 25 dB to about 32 dB, about 30 dB to about 35 dB, about 25 dB, about 27 dB, about 29 dB, about 31 dB, about 33 dB or about 35 dB, including ranges and values therebetween, for a Samsung HERA W10 or an equivalent setting on a different Ultrasound system, b. dynamic range of about 40 dB to about 45 dB, about 42 dB to about 46 dB, about 45 dB to about 50 dB, about 40 dB, about 42 dB, about 44 dB, about 46 dB, about 48 dB or about 50 dB, including ranges and values therebetween, for a Samsung HERA W10 or an equivalent setting on a different Ultrasound system, and c. mechanical index of about 0.05 to about 0.075, about 0.075 to about 0.1, about 0.1 to about 0.15, about 0.05, about 0.1 or about 0.15, including ranges and values therebetween, for a Samsung HERA W10 or an equivalent setting on a different Ultrasound system.

[0105] In some embodiments, the CEUS images are obtained from imaging performed while maintaining two or more parameters selected from: a. gain of about 25 dB to about 35 dB for a Samsung HERA W10 or an equivalent setting on a different Ultrasound system, b. dynamic range of about 40 dB to about 50 dB for a Samsung HERA W10 or an equivalent setting on a different Ultrasound system, and c. mechanical index of about 0.05 to about 0.15 for a Samsung HERA W10 or an equivalent setting on a different Ultrasound system.

[0106] In some embodiments, the CEUS images are obtained from imaging performed while maintaining a combination of the following parameters: a. gain of about 25 dB to about 35 dB for a Samsung HERA W10 or an equivalent setting on a different Ultrasound system b. dynamic range of about 40 dB to about, 50 dB for a Samsung HERA W10 or an equivalent setting on a different Ultrasound system, andc. mechanical index of about 0.05 to about 0.15 for a Samsung HERA W10 or an equivalent setting on a different Ultrasound system.

[0107] In a preferred embodiment, the CEUS images are obtained from imaging performed while maintaining one or more parameters selected from: a. gain of about 30 dB for a Samsung HERA W10 or an equivalent setting on a different Ultrasound system, b. dynamic range of about 45 dB for a Samsung HERA W10 or an equivalent setting on a different Ultrasound system, and c. mechanical index of about 0.1 for a Samsung HERA W10 or an equivalent setting on a different Ultrasound system.

[0108] In another preferred embodiment, the CEUS images are obtained from imaging performed while maintaining two or more parameters selected from: a. gain of about 30 dB for a Samsung HERA W10 or an equivalent setting on a different Ultrasound system, b. dynamic range of about 45 dB for a Samsung HERA W10 or an equivalent setting on a different Ultrasound system, and c. mechanical index of about 0.1 for a Samsung HERA W10 or an equivalent setting on a different Ultrasound system.

[0109] Accordingly, in a preferred embodiment, the method for early detection of ectopic endometrium, and preferably adenomyosis or endometriosis, comprises: using CEUS images of uterine vasculature of a subject for qualitative and quantitative analysis of enhancement patterns; wherein the CEUS images are obtained from imaging performed while maintaining one or more parameters selected from: a. gain of about 25 dB to about 35 dB for a Samsung HERA W10 or an equivalent setting on a different Ultrasound system, b. dynamic range of about 40 dB to about 50 dB for a Samsung HERA W10 or an equivalent setting on a different Ultrasound system, and c. mechanical index of about 0.05 to about 0.15 for a Samsung HERA W10 or an equivalent setting on a different Ultrasound system.[HO] In some embodiments, the method for early detection of ectopic endometrium, and preferably adenomyosis or endometriosis, comprises:using CEUS images of uterine vasculature of a subject for qualitative and quantitative analysis of enhancement patterns; wherein, preferably, the CEUS images are obtained from imaging performed while maintaining one or more parameters selected from: a. gain of about 30 dB for a Samsung HERA W10 or an equivalent setting on a different Ultrasound system, b. dynamic range of about 45 dB for a Samsung HERA W10 or an equivalent setting on a different Ultrasound system, and c. mechanical index of about 0.1 for a Samsung HERA W10 or an equivalent setting on a different Ultrasound system.[Hl] In a preferred embodiment, the method for early detection of ectopic endometrium, and preferably adenomyosis or endometriosis, is characterized by quantitative and qualitative analysis of enhancement patterns on CEUS images of uterine vasculature of a subject; wherein the detection is during the angiogenic phase, prior to onset of fibrosis; and wherein the CEUS images are obtained from imaging performed while maintaining one or more parameters selected from: a. gain of about 25 dB to about 35 dB for a Samsung HERA W10 or an equivalent setting on a different Ultrasound system, b. dynamic range of about 40 dB to about 50 dB for a Samsung HERA W10 or an equivalent setting on a different Ultrasound system, and c. mechanical index of about 0.05 to about 0.15 for a Samsung HERA W10 or an equivalent setting on a different Ultrasound system.

[0112] In some embodiment, the method for early detection of ectopic endometrium, and preferably adenomyosis or endometriosis, is characterized by quantitative and qualitative analysis of enhancement patterns on CEUS images of uterine vasculature of a subject; wherein the detection is during the angiogenic phase, prior to onset of fibrosis; and wherein, preferably, the CEUS images are obtained from imaging performed while maintaining one or more parameters selected from: a. gain of about 30 dB for a Samsung HERA W10 or an equivalent setting on a different Ultrasound system, b. dynamic range of about 45 dB for a Samsung HERA W10 or an equivalent setting on a different Ultrasound system, andc. mechanical index of about 0.1 for a Samsung HERA W10 or an equivalent setting on a different Ultrasound system.

[0113] In some embodiments, once the images are obtained, they may optionally be subjected to correction or optimization for improved clarity and to enable more accurate analysis. Without intending to be bound by theory, the optimization or correction may be performed using any suitable software in the art. In some embodiments, the image optimization may comprise one or more of motion correction, resolution enhancement, contrast optimization, adequate depth penetration, image width, spatial and temporal resolution, image contrast, artifact suppression and application of zoom.

[0114] In some embodiments, the qualitative analysis comprises visual assessment of one or more of: a. differences in vasculature between healthy and ectopic endometrial tissue; b. differences in vasculature between different uterine layers; c. signal intensity in a specific region of interest (ROI); d. homogeneity of myometrium enhancement; and e. enhancement of pelvic organs.

[0115] In some embodiments, the qualitative analysis comprises visual assessment of two, three or four of: a. differences in vasculature between healthy and ectopic endometrial tissue; b. differences in vasculature between different uterine layers; c. signal intensity in a specific region of interest (ROI); d. homogeneity of myometrium enhancement; and e. enhancement of pelvic organs.

[0116] In some embodiments, the qualitative analysis comprises visual assessment of: a. differences in vasculature between healthy and ectopic endometrial tissue; b. differences in vasculature between different uterine layers; c. signal intensity in a specific region of interest (ROI); d. homogeneity of myometrium enhancement; and e. enhancement of pelvic organs.

[0117] Accordingly, in some embodiments, the method for early detection of ectopic endometrium, preferably adenomyosis or endometriosis comprises:using CEUS images of uterine vasculature of a subject for qualitative and quantitative analysis of enhancement patterns, wherein the qualitative analysis comprises visual assessment of one, two, three four or all of: a. differences in vasculature between healthy and ectopic endometrial tissue; b. differences in vasculature between different uterine layers; c. signal intensity in a specific region of interest (ROI); d. homogeneity of myometrium enhancement; and e. enhancement of pelvic organs.

[0118] In some embodiments, the method for early detection of ectopic endometrium, and preferably adenomyosis or endometriosis, is characterized by quantitative and qualitative analysis of enhancement patterns on CEUS images of uterine vasculature of a subject; wherein the detection is during the angiogenic phase, prior to onset of fibrosis; and wherein, preferably, the qualitative analysis comprises visual assessment of one, two, three, four or all of: a. differences in vasculature between healthy and ectopic endometrial tissue; b. differences in vasculature between different uterine layers; c. signal intensity in a specific region of interest (ROI); and d. homogeneity of myometrium enhancement; and e. enhancement of pelvic organs.

[0119] In a preferred embodiment, the method for early detection of ectopic endometrium, and preferably adenomyosis or endometriosis, is characterized by quantitative and qualitative analysis of enhancement patterns on CEUS images of uterine vasculature of a subject; wherein the detection is during the angiogenic phase, prior to onset of fibrosis; wherein the qualitative analysis comprises visual assessment of one, two, three, four or all of: a. differences in vasculature between healthy and ectopic endometrial tissue; b. differences in vasculature between different uterine layers; c. signal intensity in a specific region of interest (ROI); and d. homogeneity of myometrium enhancement; and e. enhancement of pelvic organs; andwherein the CEUS images are obtained from imaging performed while maintaining one or more parameters selected from: i.gain of about 25 dB to about 35 dB for a Samsung HERA W10 or an equivalent setting on a different Ultrasound system, ii. dynamic range of about 40 dB to about 50 dB for a Samsung HERA W10 or an equivalent setting on a different Ultrasound system, and iii. mechanical index of about 0.05 to about 0.15 for a Samsung HERA W10 or an equivalent setting on a different Ultrasound system.

[0120] In some embodiments, the healthy uterine tissue is of the same or a different subject.

[0121] In some embodiments, the healthy uterine tissue may be a healthy tissue adjacent to the adenomyotic tissue.

[0122] In some embodiments, the healthy uterine tissue may be a healthy tissue adjacent to the endometriosis tissue.

[0123] In some embodiments, the healthy uterine tissue may be that of a healthy control.

[0124] In some embodiments, the visual assessment of the differences in vasculature between healthy tissue and ectopic endometrium, preferably adenomyotic or endometriosis tissue, may be performed by way of comparison of the CEUS image of the subject with a reference image of a healthy tissue.

[0125] In some embodiments, increased vascular or microvascular density may be indicative of ectopic endometrium, and preferably adenomyosis or endometriosis.

[0126] Further, in some embodiments, upon visual inspection of the CEUS scans, it may be observed that the myometrium of a healthy uterus shows homogeneous enhancement. In the patients with ectopic endometrium, preferably adenomyosis or endometriosis, the myometrium shows heterogeneous enhancement, featuring both hyper- and unenhanced spots, dependent on the presence of angiogenesis and optionally, fibrosis.

[0127] Accordingly, in some embodiments, the myometrium of a subject having ectopic endometrium, preferably adenomyosis or endometriosis shows heterogeneous enhancement. Based on the said observation with respect to homogeneity or heterogeneity of enhancement, it is possible to identify ectopic endometrium and discriminate adenomyotic or endometriosis tissue from healthy tissue and also ectopic endometriumwith or without fibrosis deceased vascularity compared to normal myometrium or compared to tissue of pelvic organs (bladder, bowel wall etc).

[0128] Thus, in some embodiments, in the qualitative analysis, the healthy uterine tissue is of the same or a different subject; and the myometrium of a subject having ectopic endometrium, preferably adenomyosis or endometriosis shows heterogeneous enhancement with enhanced or decreased angiogenesis.

[0129] The imaging and signal intensity analysis (that forms part of qualitative analysis) is usually performed on a region of interest (ROI). The ROI is a region suspected to have the tissue of interest, wherein the imaging may be performed specifically on the said region. In some embodiments, optionally, the ROI as referred to above, may be determined on the basis of corresponding B-mode ultrasound images. Accordingly, in some embodiments, optionally, the CEUS imaging may be preceded by a B-mode ultrasound to identify adenomyotic-appearing regions (i.e. regions that appear adenomyotic on B-mode), wherein the B-mode ultrasound and the CEUS imaging may be performed by the same or different devices. In some embodiments, the B-mode ultrasound may be 2D or 3D B-mode ultrasound.

[0130] Accordingly, in some embodiments, the CEUS imaging is preceded by a B-mode ultrasound to identify adenomyotic-appearing regions, wherein the B-mode ultrasound and the CEUS imaging may be performed by the same or different devices.

[0131] Thus, in some embodiments, the method identifying / detecting early stage adenomyosis comprises first determining the ROI followed by CEUS imaging of uterine vasculature in the ROI.

[0132] Therefore, in some embodiments, the method for early detection of ectopic endometrium, and preferably adenomyosis or endometriosis, comprises: a. determining ROI, preferably based on images obtained using B-mode ultrasound; and b. using CEUS images of uterine vasculature of the ROI in a subject for qualitative and quantitative analysis of enhancement patterns

[0133] In a preferred embodiment, the method for early detection of ectopic endometrium, and preferably adenomyosis or endometriosis, comprises: a. determining ROI, preferably based on images obtained using B-mode ultrasound; andb. using CEUS images of uterine vasculature of the ROI in a subject for qualitative and quantitative analysis of enhancement patterns; wherein preferably the detection is during the angiogenic phase, prior to the onset of fibrosis.

[0134] In some embodiments, the signal intensity may be further subjected to CEUS timeintensity curve (TIC) fitting. This forms part of the quantitative analysis further elaborated on below.

[0135] In some embodiments, the quantitative analysis of enhancement patterns on CEUS images of uterine vasculature of a subject comprises one or more of a. analysis of pixel-based spatial similarity using contrast-ultrasound dispersion imaging (CUDI); wherein the said analysis comprises determining one or more spatial similarity metrics selected from spectral coherence (p), correlation (r), and mutual information (I); and b. time-intensity analysis of ultrasound contrast agent (UCA) wash-in and wash-out.

[0136] In some embodiments, the quantitative analysis comprises: a. analysis of pixel-based spatial similarity using CUDI; wherein the said analysis comprises determining one or more spatial similarity metrics selected from spectral coherence (p), correlation (r), and mutual information (I); and b. time-intensity analysis of ultrasound contrast agent (UCA) wash-in and wash-out.

[0137] Accordingly, in some embodiments, the method for early detection of ectopic endometrium, and preferably adenomyosis or endometriosis, comprises: using CEUS images of uterine vasculature of a subject for qualitative and quantitative analysis of enhancement patterns, wherein the quantitative analysis comprises visual assessment of one or more of: a. analysis of pixel-based spatial similarity using CUDI; wherein the said analysis comprises determining one or more spatial similarity metrics selected from spectral coherence (p), correlation (r), and mutual information (I); and b. time-intensity analysis of ultrasound contrast agent (UCA) wash-in and wash-out.

[0138] In a preferred embodiment, the method for early detection of ectopic endometrium, and preferably adenomyosis or endometriosis, is characterized by use of CEUS images of uterine vasculature of a subject for quantitative and qualitative analysis of enhancement patterns; wherein the detection is during the angiogenic phase, prior to the onset offibrosis; and wherein the quantitative analysis comprises visual assessment of one or more of: a. analysis of pixel-based spatial similarity using CUDI; wherein the said analysis comprises determining one or more spatial similarity metrics selected from spectral coherence (p), correlation (r), and mutual information (I); and b. time-intensity analysis of ultrasound contrast agent (UCA) wash-in and wash-out.

[0139] As mentioned above, the pixel-based spatial similarity analysis is performed using the CUDI framework. In a non-limiting embodiment, in the said pixel-based spatial similarity analysis, the kernel of choice is an annulus of pixels with an inner and outer diameter (in the range of millimeters), to which the central pixel (located at the center of this annulus) is compared. In some embodiments, for quantification of spatial similarity, p, r and I are computed between the TIC of the central pixel and those of the pixels in the kernel. Without intending to be bound by theory, the performance of these spatial similarity metrics is heavily influenced by the optimization of the used annular kernel, which should be optimized such that minute changes, stemming from early angiogenic processes, can be detected.

[0140] In some embodiments, heterogeneity of p, r and I coincide with the tissue heterogeneity of ectopic endometrium, preferably adenomyosis or endometriosis, wherein lower degree of heterogeneity of the uterus is associated with a healthy uterus. In some embodiments, lower degree of heterogeneity of the bowel or bladder wall are associated with the absence of endometriosis. Accordingly, in some embodiments, the imaging of the uterus may also be coupled with parallel analysis of the bowel or bladder wall to rule out endometriosis.

[0141] To visualize results of the said analysis, parametric maps of these metrics i.e. p, r and / or I may be generated over the ROIs. The CUDI parametric maps of the spatial similarity metrics reflect the enhancement behavior, most prominently when considering the I metric.

[0142] In some embodiments, the spectral coherence (p), the correlation (r), and the mutual information (I) are inversely proportional to dispersion. Thus, in some embodiments, the higher the local r, p and / or I, the lower the local dispersion. Low dispersion is indicative of angiogenesis associated with early pathophysiology of ectopic endometrium, preferably adenomyosis or endometriosis.

[0143] In some embodiments, the time-intensity analysis of ultrasound contrast agent (UCA) wash-in and wash-out comprises monitoring the wash-in and wash-out parameters and performing a TIC fitting using the said parameters. In some embodiments, the washin of the contrast agent through the ROIs is mirrored by signal intensity parameters such as Time to peak (TTP) and Rising Time (RT). Without intending to be limited by theory, time-intensity curve analysis (TIC analysis) based on contrast-enhanced ultrasound (CEUS) provides quantifiable information about the microcirculation of different tissues. In some embodiments, the TIC fitting is performed using a suitable software. In some embodiments, the TIC software allows one to visualize the perfusion curve fitting in a graphical format, enabling parameters based on arrival and peak time of contrast.

[0144] Put together, in some embodiments, the present disclosure provides a method for early detection of ectopic endometrium, and preferably adenomyosis or endometriosis, comprising: using CEUS images of uterine vasculature of a subject for qualitative and quantitative analysis of enhancement patterns; wherein the qualitative analysis comprises assessment of one or more of microvasculature, signal intensity in a specific region of interest (ROI), and homogeneity of the myometrium enhancement; and wherein the quantitative analysis comprises pixel-based spatial similarity and timeintensity analysis of ultrasound contrast agent (UCA).

[0145] In a preferred embodiment, the method for early detection of ectopic endometrium, and preferably adenomyosis or endometriosis, is characterized by use of CEUS images of uterine vasculature of a subject for quantitative and qualitative analysis of enhancement patterns; wherein the detection is during the angiogenic phase, prior to the onset of fibrosis; wherein the qualitative analysis comprises assessment of one or more of microvasculature, signal intensity in a specific region of interest (ROI), and homogeneity of the myometrium enhancement; and wherein the quantitative analysis comprises pixel-based spatial similarity and timeintensity analysis of ultrasound contrast agent (UCA).

[0146] In some embodiments, the method for early detection of ectopic endometrium, and preferably adenomyosis or endometriosis is characterized by use of CEUS images of uterine vasculature of a subject for quantitative and qualitative analysis of enhancementpatterns; wherein the detection is during the angiogenic phase, prior to the onset of fibrosis; wherein, preferably, the qualitative analysis comprises assessment of one or more of microvasculature, signal intensity in a specific region of interest (ROI), and homogeneity of the myometrium enhancement; and wherein, preferably, the quantitative analysis comprises pixel-based spatial similarity and time-intensity analysis of ultrasound contrast agent (UCA).

[0147] In a preferred embodiment, the method for early detection of ectopic endometrium, and preferably adenomyosis or endometriosis, is characterized by use of CEUS images of uterine vasculature of a subject for quantitative and qualitative analysis of enhancement patterns; wherein the detection is during the angiogenic phase, prior to the onset of fibrosis; wherein the qualitative analysis comprises assessment of one or more of microvasculature, signal intensity in a specific region of interest (ROI), and homogeneity of the myometrium enhancement; wherein the quantitative analysis comprises pixel-based spatial similarity and timeintensity analysis of ultrasound contrast agent (UCA); and wherein the CEUS images are obtained from imaging performed while maintaining one or more parameters selected from: i.gain of about 25 dB to about 35 dB for a Samsung HERA W10 or an equivalent setting on a different Ultrasound system, ii. dynamic range of about 40 dB to about 50 dB for a Samsung HERA W10 or an equivalent setting on a different Ultrasound system, and iii. mechanical index of about 0.05 to about 0.15 for a Samsung HERA W10 or an equivalent setting on a different Ultrasound system.

[0148] In some embodiments, the method for early detection of ectopic endometrium, and preferably adenomyosis or endometriosis, is characterized by use of CEUS images of uterine vasculature of a subject for quantitative and qualitative analysis of enhancement patterns; wherein the detection is during the angiogenic phase, prior to the onset of fibrosis; wherein, preferably, the quantitative analysis comprises visual assessment of one or more of:a. analysis of pixel-based spatial similarity using CUDI; wherein the said analysis comprises determining one or more spatial similarity metrics selected from spectral coherence (p), correlation (r), and mutual information (I); and b. time-intensity analysis of ultrasound contrast agent (UCA) wash-in and wash-out; and wherein, preferably, the qualitative analysis comprises visual assessment of one, two, three, four or all of: a. differences in vasculature between healthy and ectopic endometrial tissue; b. differences in vasculature between different uterine layers; c. signal intensity in a specific region of interest (ROI); and d. homogeneity of myometrium enhancement; and enhancement of pelvic organs.

[0149] In another preferred embodiment, the method for early detection of ectopic endometrium, and preferably adenomyosis or endometriosis, is characterized by use of CEUS images of uterine vasculature of a subject for quantitative and qualitative analysis of enhancement patterns; wherein the detection is during the angiogenic phase, prior to the onset of fibrosis; wherein the quantitative analysis comprises visual assessment of one or more of: a. analysis of pixel-based spatial similarity using CUDI; wherein the said analysis comprises determining one or more spatial similarity metrics selected from spectral coherence (p), correlation (r), and mutual information (I); and b. time-intensity analysis of ultrasound contrast agent (UCA) wash-in and wash-out; wherein the qualitative analysis comprises visual assessment of one, two, three, four or all of: a. differences in vasculature between healthy and ectopic endometrial tissue; b. differences in vasculature between different uterine layers; c. signal intensity in a specific region of interest (ROI); and d. homogeneity of myometrium enhancement; and e. enhancement of pelvic organs; and wherein, preferably, the CEUS images are obtained from imaging performed while maintaining one or more parameters selected from:i. gain of about 25 dB to about 35 dB for a Samsung HERA W10 or an equivalent setting on a different Ultrasound system, ii. dynamic range of about 40 dB to about 50 dB for a Samsung HERA W10 or an equivalent setting on a different Ultrasound system, and iii. mechanical index of about 0.05 to about 0.15 for a Samsung HERA W10 or an equivalent setting on a different Ultrasound system.Method of differentiating between different stages of adenomyosis or endometriosis

[0150] Further provided in the present disclosure is a method of differentiating between different stages of severity or progression of disorders associated characterized by ectopic endometrium comprising using CEUS images of uterine vascular structures of a subject and comparing variation in CEUS enhancement patterns between healthy and ectopic endometrial tissues.

[0151] In some embodiments, the present disclosure provides a method of differentiating between different stages of adenomyosis or endometriosis comprising using CEUS images of uterine vascular structures of a subject and comparing variation in CEUS enhancement patterns between healthy and adenomytic or endometriosis tissues.

[0152] In some embodiments, provided herein is a method of differentiating between early and advanced stage adenomyosis or endometriosis comprising using CEUS images of uterine vascular structures of a subject and comparing variation in CEUS enhancement patterns between healthy and adenomyotic tissues or endometriosis tissues.

[0153] In some embodiments, early stage adenomyosis or endometriosis is characterized by ectopic endometrium with enhanced angiogenesis compared to normal myometrium, without fibrosis. Accordingly, in a preferred embodiment, provided herein is a method of differentiating between early and later stages of adenomyosis or endometriosis, comprising using CEUS images of uterine vascular structures of a subject and comparing variation in CEUS enhancement patterns between healthy and adenomytic or endometriosis tissues; wherein the early stage of adenomyosis or endometriosis is at an angiogenic phase, prior to onset of fibrosis.

[0154] In some embodiments, advanced adenomyosis or endometriosis tissue is characterized by ectopic endometrium tissue with decreased angiogenesis compared to normal myometrium or pelvic tissue.

[0155] In order to ascertain the difference between early and late stage adenomysosis or endometriosis, in some embodiments, the CEUS imaging and CUDI based analysis may be additionally coupled with elastography which may be used to check tissue stiffness. Elastography allows non-invasive measurement of tissue stiffness. Stiff areas in the uterus are a sign of scar tissue (fibrosis), indicative of late stage adenomyosis or endometriosis. Thus, in a preferred embodiment, the method of differentiating between early and advanced stage adenomyosis or endometriosis further comprises using elastography images of the uterine vascular structures to identify presence or absence of fibrosis.

[0156] Accordingly, in some embodiments, the method of differentiating between early and advanced stage adenomyosis or endometriosis comprises: a. using CEUS images of uterine vasculature of a subject for qualitative and comparing variation in CEUS enhancement or decrease patterns between healthy and adenomyotic or endometriosis tissue; and optionally, b. using elastography images of the uterine vascular structures to identify presence or absence of fibrosis.

[0157] In some embodiments, the method of differentiating between early and advanced stage adenomyosis or endometriosis comprises: a. using CEUS images of uterine vasculature of a subject for qualitative and comparing variation in CEUS enhancement or decrease patterns between healthy and adenomyotic or endometriosis tissue; and b. using elastography images of the uterine vascular structures to identify presence or absence of fibrosis.

[0158] Put together, using CEUS and CUDI analyses as described herein, it is possible to differentiate between 1) normal uterus, 2) enhanced angiogenesis in the inner myometrium layer (junctional zone), 3) enhanced angiogenesis in middle myometrium, 4) enhanced angiogenesis in the outer myometrium compared to normal myometrium 5) advanced stage adenomyosis is characterized by decreased angiogenesis in comparison to normal myometrium and 6) presence or absence of early endometriosis with enhanced angiogenesis or late endometriosis with decreased angiogenesis.Stand-alone system for off-line analysis

[0159] Further envisaged in the present disclosure is a system for facilitating the method(s) of the present disclosure as described above.

[0160] In some embodiments, the system is a stand-alone system that facilitates off-line analysis of a subject or a CEUS image of the said subject.

[0161] In some embodiments, provided herein is a system for early detection of ectopic endometrium, and preferably adenomyosis or endometriosis, that comprises one or more imaging devices programmed to operate at optimized parameters to facilitate one or more of: a. B-mode ultrasound; b. CEUS; and c. CUDI based analysis of the CEUS images.

[0162] In some embodiments, the said system is further equipped with software to facilitate analysis of results of the one or more of B-mode ultrasound, CEUS and CUDI based analysis of the CEUS images to facilitate early detection of adenomyosis.

[0163] In some embodiments, the system for early detection of ectopic endometrium, and preferably adenomyosis or endometriosis, may solely comprise a software into which the CEUS images may be fed to obtain a qualitative and quantitative analysis of the said images. In some embodiments, the said system may comprise: a. Software for qualitative analysis of the CEUS images, b. CUDI framework for quantitative analysis of the CEUS images, and c. Optionally, pre-fed reference data to facilitate comparison versus healthy control.Treatment module

[0164] Further envisaged herein is a non-steroidal angiogenesis inhibitor for use in the treatment or prevention of early stage adenomyosis or early stage endometriosis in a patient diagnosed by the method as described above.

[0165] An angiogenesis inhibitor is a substance that inhibits the growth of new blood vessels (angiogenesis). Angiogenesis inhibitors have been closely studied for possible cancer treatments. Studies on cancer usually use the highest possible dose, to reach the highest effect. “Bombing” the tumor with angiogenesis inhibitors may be acting more as chemotherapy, masking the specific anti-angiogenesis effect. Furthermore, malignant cells have evolved an angiogenic response that is different from physiological angiogenesis, which is still responsive to intervention. Additionally, tumor cells are genetically instable and keep mutating towards therapy-resistant variants. As endometrial cells are benign andgenetically stable, therapy resistance is less likely to occur. It is for these reasons that anti- angiogenic intervention in benign uterine disorders such as adenomyosis is expected to be successful and may lead to the development of an efficient non-hormonal local treatment.

[0166] While not wishing to be bound by theory, it is proposed herein that treatment with an angiogenesis inhibitor reduces the growth of endometrial tissue in the muscular wall of the uterus, resulting in less adenomyosis and / or the corresponding symptoms. The present disclosure proposes that the therapeutic compounds disclosed herein affect the angiogenesis of the endometrial tissue. Inhibition of angiogenesis would result in suppressed myometrial infiltration by endometrial tissue and thus alleviation of symptoms.

[0167] In some embodiments, the non-steroidal angiogenesis inhibitor is administered intra-uterine.

[0168] In some embodiments, the intrauterine administration is provided by an intrauterine device (IUD). The IUD may slowly release the angiogenesis inhibitor. Preferably, the IUD releases the inhibitor over the course of several months, preferably over the course of several years. In this way the inhibitor is provided for slow / extended release. In some embodiments, the non-steroidal angiogenesis inhibitor is a small molecule or an antigen binding molecule such as an antibody or antigen binding fragment thereof.

[0169] In some embodiments, the non-steroidal angiogenesis inhibitor targets an angiogenic signaling axis, e.g. VEGF, VEGF receptor, EGF, EGF receptor, PDGF, PDGF receptor, or PGF, PGF receptor or an angiogenesis-associated molecule or receptor, e.g. an integrin, CD36, CD44, extracellular vimentin, flbrillin-2, secreted frizzled-related protein-2, lysyl oxidase, prostate specific membrane antigen, versican, apelin.

[0170] In some embodiments, the non-steroidal angiogenesis inhibitor is an anti-VEGF binding molecule such as an antibody or antigen binding fragment thereof, a peptibody or a nanobody; wherein preferably, the angiogenesis inhibitor is selected from bevacizumab, ramucirumab, or ranibizumab.

[0171] In some embodiments, the non-steroidal angiogenesis inhibitor is a tyrosine kinase inhibitor, preferably selected from axitinib, imatinib, erlotinib, cabozantinib, lapatinib, pazopanib, ponatinib, regorafenib, sunitinib, sorafenib, vandetanib, and crizotinib and / or a pharmaceutically acceptable salt thereof.Advantages o f the present disclosure

[0172] Uterine CEUS is a feasible technique to image vascular structures in the uterus to identify and characterize ectopic endometrium, preferably adenomyosis or endometriosis. The results from the TIC and CUDI spatial similarity allow accurate characterization and discrimination of ectopic endometrial tissue from healthy tissue.

[0173] The method of the present disclosure allows early detection of adenomyosis or endometriosis.

[0174] The method, further, helps distinguish between early and late stages of adenomyosis or endometriosis.

[0175] The technical implementation of the method of the present disclosure is straightforward, requiring only a software upgrade of existing scanners for dedicated imaging schemes. No costly hardware modification is required.

[0176] The implementation of a stand-alone system for off-line analysis as described above allows easy application of the method of the present disclosure.

[0177] The subject matter of embodiments of the present invention is described here with specificity to meet statutory requirements, but this description is not necessarily intended to limit the scope of the disclosure. The disclosed subject matter may be embodied in other ways, may include different elements or steps, and may be used in conjunction with other existing or future technologies. This description should not be interpreted as implying any particular order or arrangement among or between various steps or elements except when the order of individual steps or arrangement of elements is explicitly described.EXAMPLESEXAMPLE 1:

[0178] Subjects: Adult women (n=15) visiting the tertiary referral Uterine Repair Center, Amsterdam UMC, presenting with prominent sonographic features of adenomyosis, e.g., hyperechoic islands and myometrial cysts, and a healthy control (n=l) were included in the study.

[0179] Imaging: After a standard B-mode scan, the subjects underwent a two-dimensional (2D) CEUS examination by bolus-tracking of SonoVueTM (Bracco, Geneva) with a HERA W10 (Samsung Medison, Republic of Korea) ultrasound system, using an endovaginal EV3-10A or EV2-10B probe. Machine settings, such as gain, dynamic range and mechanical index, were varied across patients in order to optimize theacquisition for uterine CEUS. A custom transvaginal probe fixture was used to minimize uterine motion during the five-minute recordings.

[0180] Analysis: Enhancement patterns of the resulting scans were analyzed qualitatively and quantitatively. Firstly, signal intensity of the discernible structures relative to each other, as well as the homogeneity of the myometrium enhancement, were visually assessed. Secondly, time-intensity analysis of the ultrasound contrast agent wash-in and wash-out was performed using VueBox™ (Bracco, Geneva). In VueBox™, regions-of- interest (ROIs) were created for the uterine corpus, adenomyotic-appearing regions (i.e. regions that appeared adenomyotic on B-mode), adjacent healthy-appearing myometrium and endometrium on the 2D CEUS images based on the corresponding 2D B-mode images. VueBox™ proprietary motion correction was applied to the scans. Time-intensity curves (TIC) were fitted to the measured CEUS intensities and wash-in (i.e. rise time and time-to-peak) and wash-out time parameters (i.e. fall time) were extracted.

[0181] Pixel-based spatial similarity analysis was performed using CUDI framework. In this analysis, the kernel of choice was an annulus of pixels with an inner and outer diameter (in the range of millimeters), to which the central pixel (i.e., located at the center of this annulus) was compared. For quantification of spatial similarity, p, r and I were computed between the TIC of the central pixel and those of the pixels in the kernel. To visualize these results, parametric maps of these metrics were generated over the ROIs.

[0182] Results: Fifteen adenomyosis patients and one healthy control were included in this pilot study, of which ten adenomyosis patients and the healthy control could be quantitatively analyzed. It was found that uterine CEUS was straightforward to perform and well-accepted by all patients. The optimal settings for 2D CEUS were found to be a gain of 30 dB, dynamic range of 45 dB, and mechanical index of 0.1. Upon visual inspection of the CEUS scans, it was seen that the myometrium of the healthy control was homogeneously enhanced and the endometrium was hypo-enhanced. In the adenomyosis patients, the myometrium that appeared adenomyotic on B-mode showed heterogeneous enhancement, featuring both hyper- and unenhanced spots and the endometrium was hypo- enhanced. There was a large variation in CEUS enhancement patterns across patients (Fig. 1).

[0183] Based on VueBox™ analysis, wash-in and wash-out times were extended in adenomyotic-appearing regions when compared to adjacent healthy-appearing myometrium and to the myometrium of the healthy control. The p, r and I all showedhigher values and a high heterogeneity in the adenomyotic myometrium, hence associated with heterogeneity in the local dispersion. The heterogeneity of p, r and I were found to coincide with the tissue heterogeneity of adenomyosis, hence they are potentially relevant features and warrant further investigation. Representative CUDI parametric maps of an adenomyotic uterus (different stages) and a healthy uterus, for comparison, are provided in Figs. 2-5, which depict expected lower degree of heterogeneity in the healthy uterus, presented through all spatial similarity metrics.

[0184] Additional embodiments and features of the present disclosure will be apparent to one of ordinary skill in art based on the description provided herein. The embodiments herein provide various features and advantageous details thereof in the description. Descriptions of well-known / conventional methods and techniques are omitted so as to not unnecessarily obscure the embodiments herein.

[0185] The foregoing description fully reveals the general nature of the embodiments herein that others can, by applying current knowledge, readily modify and / or adapt for various applications such specific embodiments without departing from the generic concept, and, therefore, such adaptations and modifications should and are intended to be comprehended within the meaning and range of equivalents of the disclosed embodiments. It is to be understood that the phraseology or terminology employed herein is for the purpose of description and not of limitation. Therefore, while the embodiments in this disclosure have been described in terms of preferred embodiments, those skilled in the art will recognize that the embodiments herein can be practiced with modification within the spirit and scope of the embodiments as described herein, without departing from the principles of the disclosure.

[0186] Any discussion of documents, acts, materials, devices, articles and the like that has been included in this specification is solely for the purpose of providing a context for the disclosure. It is not to be taken as an admission that any or all of these matters form a part of the prior art base or were common general knowledge in the field relevant to the disclosure as it existed anywhere before the priority date of this application.References:1. Qualitative and quantitative features of deep endometriosis in contrast-enhanced ultrasound: An initial experience and literature review; Zhang et al., Clinical Hemorheology and Microcirculation 85 (202.3) 7.3-82.2. Spatiotemporal Correlation of Ultrasound Contrast Agent Dilution Curves for Angiogenesis Localization by Dispersion Imaging; Maarten P. J. Kuenen; Transactions on Ultrasonics, Ferroelectrics, And Frequency Control, Vol. 60, No. 12, December 2013.

Claims

CLAIMS:

1. A method for early detection of ectopic endometrium, characterized by quantitative and qualitative analysis of enhancement patterns on contrast-enhanced ultrasound (CEUS) images of uterine vasculature of a subject; wherein the detection is during the angiogenic phase, prior to onset of fibrosis.

2. The method according to claim 1, wherein the early detection of ectopic endometrium is indicative of adenomyosis or endometriosis.

3. The method according to any of claims 1-2, wherein the quantitative analysis comprises one or more of: a. analysis of pixel-based spatial similarity using contrast-ultrasound dispersion imaging (CUDI); wherein the said analysis comprises determining one or more spatial similarity metrics selected from spectral coherence (p), correlation (r), and mutual information (I); and b. time-intensity analysis of ultrasound contrast agent (UCA) wash-in and washout.

4. The method according to claim 3, wherein the spectral coherence (p), the correlation (r), and the mutual information (I) are inversely proportional to dispersion; and wherein low dispersion is indicative of angiogenesis associated with early pathophysiology of ectopic endometrium, preferably adenomyosis or endometriosis.

5. The method according to any of claims 1-4, wherein the qualitative analysis comprises visual assessment of one or more of: a. differences in vasculature between healthy and ectopic endometrial tissue; b. differences in vasculature between different uterine layers; c. signal intensity in a specific region of interest (ROI); and d. homogeneity of myometrium enhancement; and e. enhancement of pelvic organs.

6. The method according to claim 5, wherein the healthy uterine tissue is of the same or a different subject; and the myometrium of a subject having ectopic endometrium shows heterogeneous enhancement.

7. The method according to claim 5, wherein the signal intensity is subjected to CEUS time-intensity curve fitting.

8. A method of differentiating between early and later stages of adenomyosis or endometriosis comprising using CEUS images of uterine vascular structures of asubject and comparing variation in CEUS enhancement patterns between healthy and adenomytic or endometriosis tissues; wherein the early stage of adenomyosis or endometriosis is at an angiogenic phase, prior to onset of fibrosis.

9. The method according to any of claims 1-8, wherein the early stage adenomyosis or endometriosis is characterized by ectopic endometrium with enhanced angiogenesis compared to normal myometrium, without fibrosis.

10. The method according to any of claims 8-9, wherein the method further comprises using elastography images of the uterine vascular structures to identify presence or absence of fibrosis.

11. The method according to any of claims 1-10, wherein the CEUS images are obtained from CEUS optimized for uterine vasculature.

12. The method according to any of claims 1-11, wherein the CEUS images are obtained from CEUS imaging performed while maintaining one or more parameters selected from: gain of about 25 dB to about 35dB, dynamic range of about 40 dB to about 50 dB, and mechanical index of about 0.01 to about 0.15.

13. A non-steroidal angiogenesis inhibitor for use in the treatment or prevention of early stage adenomyosis or early stage endometriosis in a patient diagnosed by the method of any of claims 1-12.

14. The non-steroidal angiogenesis inhibitor for use according to claim 13, wherein the non-steroidal angiogenesis inhibitor is administered intra-uterine.

15. The non-steroidal angiogenesis inhibitor for use according to any of claims 13-14, wherein the angiogenesis inhibitor is a small molecule or an antigen binding molecule such as an antibody or antigen binding fragment thereof.

16. The non-steroidal angiogenesis inhibitor for use according to any of claims 13-15, wherein the angiogenesis inhibitor targets an angiogenic signaling axis, e.g. VEGF, VEGF receptor, EGF, EGF receptor, PDGF, PDGF receptor, or PGF, PGF receptor or an angiogenesis-associated molecule or receptor, e.g. an integrin, CD36, CD44, extracellular vimentin, fibrillin-2, secreted frizzled-related protein-2, lysyl oxidase, prostate specific membrane antigen, versican, apelin.

17. The non-steroidal angiogenesis inhibitor for use according to any of claims 13-16, wherein the angiogenesis inhibitor is an anti-VEGF binding molecule such as an antibody or antigen binding fragment thereof, a peptibody or a nanobody; whereinpreferably, the angiogenesis inhibitor is selected from bevacizumab, ramucirumab, or ranibizumab.

18. The non-steroidal angiogenesis inhibitor for use according to claim 15, wherein the small molecule is a tyrosine kinase inhibitor, preferably selected from axitinib, imatinib, erlotinib, cabozantinib, lapatinib, pazopanib, ponatinib, regorafenib, sunitinib, sorafenib, vandetanib, and crizotinib and / or a pharmaceutically acceptable salt thereof.

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