The present disclosure relates generally to gynecological malignancies. More specifically, the disclosure is directed to method, device and kit for in vitro screening of endometrial cancer, cervical cancer, or fallopian tube cancer using menstrual blood or vaginal discharge.
BACKGROUND OF THE INVENTION
[0002] Background description includes information that may be useful in understanding the present invention. It is not an admission that any of the information provided herein is prior art or relevant to the presently claimed invention, or that any publication specifically or implicitly referenced is prior art. [0003] Gynecological cancer is a cancer that arises from the female reproductive tract. Cancer that arises from uterine endometrium is called endometrial cancer or Carcinoma endometrium. Carcinoma endometrium is the most common gynecological cancer in developed countries, with an age-standardized incidence rate (world) of 8.4 per 100,000 women. In developing countries, cervical cancer remains the leading gynecological cancer, but recently there has been an increase in endometrial cancer incidence. In India, the total number of estimated new endometrial cancer cases in 2018 was 13,328, with an estimated 5010 deaths. The age-standardized incidence rate (ASIR) of endometrial cancer in India is 2.3/100,000 women. The rise in endometrial cancer in India is mainly attributed to changing trends in women's lifestyle and reproductive profile, especially in urban areas (ICMR). Cancer arising from the cervix is called cervical cancer. India has a population of 436.76 million women aged 15 years and older who are at risk of developing cervical cancer. As per estimations for 2012, every year 122844 women are diagnosed with cervical cancer, and 67477 die from the disease. In India, cervical cancer is the second most common cancer among women of age 15 to 44 (National Health portal). Cancer arising from the fallopian tube is termed fallopian tube cancer. It is a comparatively rare form of gynecological cancer with an incidence of about 1 in 100.

[0004] Major problems faced in detecting female gynecological cancer in India are ignorance of symptoms, shyness, lack of health infrastructure to detect disease in the early stages, and affordability of the available methods to detect cancer in an early stage. Additionally, the existing techniques such as biopsy are invasive further lowering patient compliance.
[0005] Thus, there is a need in the art for developing new non-invasive methods, devices and kits for early screening of gynecological cancer.
OBJECTS OF THE INVENTION
[0006] An object of the present disclosure is to provide an in vitro method of
screening endometrial cancer, cervical cancer, or fallopian tube cancer.
[0007] An object of the present disclosure is to provide an in vitro method of
screening endometrial cancer, cervical cancer, or fallopian tube cancer using
menstrual blood or vaginal discharge.
[0008] An object of the present disclosure is to provide a device for collecting a
sample for in vitro screening of endometrial cancer, cervical cancer, or fallopian
tube cancer.
[0009] Another object of the present disclosure is to provide a kit for screening
endometrial cancer, cervical cancer, or fallopian tube cancer in a sample.
SUMMARY OF THE INVENTION
[0010] This summary is provided to introduce a selection of concepts in a simplified form that are further described below in Detailed Description section. This summary is not intended to identify key features or essential features of the claimed subject matter, nor is it intended to be used as an aid in determining the scope of the claimed subject matter.
[0011] Secretory proteins play essential roles in the cross-talk of individual functional units, including cells. Since secretory proteins are critical for signal transduction, they are closely related to disease development, including malignant, metabolic, and neural diseases. The present disclosure provides a method, device

and kit for in vitro screening of endometrial cancer, cervical cancer, or fallopian tube cancer using secretory proteins in menstrual blood or vaginal discharges. [0012] Aspects of the present disclosure rely on the secretion of specific protein biomarkers by malignant cells in endometrial cancer, cervical cancer, or fallopian tube cancer in menstrual blood or vaginal discharge.
[0013] In an aspect, the present disclosure provides an in vitro method of screening one or more protein biomarkers associated with endometrial cancer, cervical cancer, or fallopian tube cancer in a sample, wherein the method comprises the steps of: (a) culturing the sample; (b) separating the protein biomarkers from the sample; (c) analyzing the protein biomarkers in the sample by mass-spectrometry to give a protein biomarker profile of the sample; and (d) comparing the protein biomarker profile with a reference protein biomarker profile; wherein the sample is menstrual blood or vaginal discharge. [0014] In an aspect, the present disclosure provides a device for collecting a sample for in vitro screening of endometrial cancer, cervical cancer, or fallopian tube cancer; wherein the device comprises: a centre collection point configured to receive the sample and has a high abundance of the sample; and a circular blade emerging from the centre collection point and concentrically oriented around the centre collection point and is configured to gradually distribute the sample away from the centre collection point; wherein the sample is menstrual blood or vaginal discharge.
[0015] In an aspect, the present disclosure provides a kit for detecting one or more protein biomarkers associated with endometrial cancer, cervical cancer, or fallopian tube cancer in a sample comprising: (a) a sample pad for receiving the sample; (b) a conjugate area downstream of the sample pad comprising one or more antibodies that bind specifically to the protein biomarkers to form complexes; (c) one or more test line(s) downstream of the conjugate area comprising labeled reagents for capturing the complexes; and (d) a control line downstream of the test lines that indicates completion of assay.

[0016] Other aspects of the invention will be set forth in the description which follows, and in part will be apparent from the description, or may be learnt by the practice of the invention.
BRIEF DESCRIPTION OF THE DRAWINGS
[0017] The following drawings form part of the present specification and are included to further illustrate aspects of the present disclosure. The disclosure may be better understood by reference to the drawings in combination with the detailed description of the specific embodiments presented herein.
[0018] Figure 1 provides an illustration of an exemplary embodiment of the device for collecting a sample.
DETAILED DESCRIPTION OF THE INVENTION
[0019] The following is a detailed description of embodiments of the disclosure. The embodiments are in such detail as to clearly communicate the disclosure. However, the amount of detail offered is not intended to limit the anticipated variations of embodiments; on the contrary, the intention is to cover all modifications, equivalents, and alternatives falling within the spirit and scope of the present disclosure as defined by the appended claims.
[0020] All publications herein are incorporated by reference to the same extent as if each individual publication or patent application were specifically and individually indicated to be incorporated by reference. Where a definition or use of a term in an incorporated reference is inconsistent or contrary to the definition of that term provided herein, the definition of that term provided herein applies and the definition of that term in the reference does not apply. [0021] Reference throughout this specification to "one embodiment" or "an embodiment" means that a particular feature, structure or characteristic described in connection with the embodiment is included in at least one embodiment. Thus, the appearances of the phrases "in one embodiment" or "in an embodiment" in various places throughout this specification are not necessarily all referring to the same embodiment. Furthermore, the particular features, structures, or

characteristics may be combined in any suitable manner in one or more embodiments.
[0022] In some embodiments, numbers have been used for quantifying weights, volumes, percentages, and so forth, to describe and claim certain embodiments of the invention and are to be understood as being modified in some instances by the term "about." Accordingly, in some embodiments, the numerical parameters set forth in the written description and attached claims are approximations that can vary depending upon the desired properties sought to be obtained by a particular embodiment. In some embodiments, the numerical parameters should be construed in light of the number of reported significant digits and by applying ordinary rounding techniques. Notwithstanding that the numerical ranges and parameters setting forth the broad scope of some embodiments of the invention are approximations, the numerical values set forth in the specific examples are reported as precisely as practicable. The numerical values presented in some embodiments of the invention may contain certain errors necessarily resulting from the standard deviation found in their respective testing measurements. [0023] Various terms as used herein are shown below. To the extent a term used in a claim is not defined below, it should be given the broadest definition persons in the pertinent art have given that term as reflected in printed publications and issued patents at the time of filing.
[0024] As used in the description herein and throughout the claims that follow, the meaning of "a," "an," and "the" includes plural reference unless the context clearly dictates otherwise. Also, as used in the description herein, the meaning of "in" includes "in" and "on" unless the context clearly dictates otherwise. [0025] Unless the context requires otherwise, throughout the specification which follow, the word "comprise" and variations thereof, such as, "comprises" and "comprising" are to be construed in an open, inclusive sense that is as "including, but not limited to."
[0026] The recitation of ranges of values herein is merely intended to serve as a shorthand method of referring individually to each separate value falling within

the range. Unless otherwise indicated herein, each individual value is incorporated into the specification as if it were individually recited herein. [0027] All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g. "such as") provided with respect to certain embodiments herein is intended merely to better illuminate the invention and does not pose a limitation on the scope of the invention otherwise claimed. No language in the specification should be construed as indicating any non-claimed element essential to the practice of the invention. [0028] Groupings of alternative elements or embodiments of the invention disclosed herein are not to be construed as limitations. Each group member can be referred to and claimed individually or in any combination with other members of the group or other elements found herein. One or more members of a group can be included in, or deleted from, a group for reasons of convenience and/or patentability. When any such inclusion or deletion occurs, the specification is herein deemed to contain the group as modified.
[0029] The description that follows, and the embodiments described therein, is provided by way of illustration of an example, or examples, of particular embodiments of the principles and aspects of the present disclosure. These examples are provided for the purposes of explanation, and not of limitation, of those principles and of the disclosure.
[0030] It should also be appreciated that the present disclosure can be implemented in numerous ways, including as a system, a method or a device. In this specification, these implementations, or any other form that the invention may take, may be referred to as processes. In general, the order of the steps of the disclosed processes may be altered within the scope of the invention. [0031] The headings and abstract of the invention provided herein are for convenience only and do not interpret the scope or meaning of the embodiments. [0032] The following discussion provides many example embodiments of the inventive subject matter. Although each embodiment represents a single combination of inventive elements, the inventive subject matter is considered to

include all possible combinations of the disclosed elements. Thus if one
embodiment comprises elements A, B, and C, and a second embodiment
comprises elements B and D, then the inventive subject matter is also considered
to include other remaining combinations of A, B, C, or D, even if not explicitly
disclosed.
[0033] As used herein the terms 'secretory protein(s)' and 'protein biomarker(s)'
have been used interchangeably.
[0034] The term, "subject" as used herein refers to an animal, preferably a
mammal of female gender and most preferably a human. The term "mammal"
used herein refers to warm-blooded vertebrate animals of the class 'mammalia',
including humans, the term mammal includes animals such as cat, dog, rabbit,
bear, fox, wolf, monkey, deer, mouse, pig and human.
[0035] Aspects of the present disclosure rely on the secretion of specific protein
biomarkers by malignant cells in endometrial cancer, cervical cancer, or fallopian
tube cancer in menstrual blood or vaginal discharge. Malignant cells secrete more
proteins, which can be potential biomarkers in protein modifications, mutations,
and concentration changes.
[0036] In an embodiment, the present disclosure provides an in vitro method of
screening one or more protein biomarkers associated with endometrial cancer,
cervical cancer, or fallopian tube cancer in a sample, wherein the method
comprises the steps of: (a) culturing the sample; (b) separating the protein
biomarkers from the sample; (c) analyzing the protein biomarkers in the sample
by mass-spectrometry to give a protein biomarker profile of the sample; and (d)
comparing the protein biomarker profile with a reference protein biomarker
profile; wherein the sample is menstrual blood or vaginal discharge.
[0037] In an embodiment, the sample may be obtained from a subject using any
blood sampling device or technique. In a preferred embodiment, the sample may
be obtained using the device for collecting a sample as disclosed herein.
[0038] In an embodiment, culturing the sample involves culturing cells in the
sample in a suitable media. The media may be comprise amino acids, vitamins,

inorganic salts, glucose, growth factors, hormones, buffer, or combinations
thereof.
[0039] In an embodiment, the protein biomarkers may be separated from the
sample by liquid chromatography. In some embodiments, the liquid
chromatography separates and quantifies the amount of each protein biomarker
present in the sample.
[0040] In an embodiment, the mass-spectrometry (MS) analysis provides a
detailed protein biomarker profiling of the sample which is assessed to detect any
shift or modification in the protein biomarkers. Mass spectrometry-based
secretome profiling is a powerful strategy to identify and characterize secretory
proteins. Proteins may be identified based on their masses. The analysis also
confirms any mutations or post-translational modifications in the protein
biomarkers of the sample. Post-translational modifications of any protein may be
detected using MS peptide sequencing.
[0041] In an embodiment, a differential value of the protein biomarker profile and
the reference protein biomarker profile maybe indicative of endometrial cancer,
cervical cancer, or fallopian tube cancer.
[0042] In an embodiment, comparing the protein biomarker profile of the sample
with reference protein biomarker profile gives an estimate of modification or
mutation in the protein biomarker. In some embodiments, a comparison is also
made in the quantities of each of the protein biomarkers in the sample and the
reference protein biomarker profile.
[0043] In an embodiment, comparing the protein biomarker profile with reference
protein biomarker profile may be performed via computational analysis. In an
embodiment, the protein biomarker profile of the sample may be stored in a
database for access at later stage.
[0044] In an embodiment, the sample may be obtained after cleaning the vaginal
area. In an embodiment, the cleaning may be performed by water or an intimate
mild antiseptic wash solution. In an embodiment, the menstrual blood may be
collected during initial days of menstrual cycle.

[0045] The method is a non-invasive way of detection of female genital tract
malignancies. In an embodiment, the method is a non-invasive biopsy for
menstruating women. In an embodiment, the method improves patient
compliance.
[0046] In an embodiment, the method may be suitable for an early detection of
cancer as well as chronic condition of cancer. In a preferred embodiment, the
method may be suitable for early detection of cancer when the subject may be
asymptomatic.
[0047] In an embodiment, protein biomarker profiles of a subject may be recorded
over a period of time. In an embodiment, the method provides a personalized and
routine screening technique. In an embodiment, the sample may be tested monthly
based on the menstrual cycle.
[0048] In an embodiment, the method is simple, reliable and robust. In an
embodiment, the method can improve timely detection and subsequent treatment
of a subject.
[0049] In an embodiment, the method, along with other clinical tests, aids any
healthcare provider in making an informed decision. In an embodiment, the
method reduces unnecessary invasive techniques of detection such as biopsy.
[0050] In an embodiment, the present disclosure provides a device for collecting a
sample for in vitro screening of endometrial cancer, cervical cancer, or fallopian
tube cancer; wherein the device comprises: a centre collection point configured to
receive the sample and has a high abundance of the sample; and a circular blade
emerging from the centre collection point and concentrically oriented around the
centre collection point and is configured to gradually distribute the sample away
from the centre collection point; wherein the sample is menstrual blood or vaginal
discharge.
[0051] In an embodiment, the circular blade has a gradual decrease in width along
its length. In an embodiment, the circular blade has a gradual decrease in
abundance of the sample along its length. In an embodiment, the circular blade
has a cleaner, simpler sample with lower abundance of protein biomarkers.

[0052] In an embodiment, the sample from the device may be extracted for use in
the in vitro method of screening endometrial cancer, cervical cancer, or fallopian
tube cancer.
[0053] In an embodiment, the device may be sterilized before use. In an
embodiment, the device may be disposed after each use.
[0054] Figure 1 provides an illustration of an exemplary embodiment of the
device for collecting a sample. The device (100) for collecting a sample for in
vitro screening of endometrial cancer, cervical cancer, or fallopian tube cancer;
comprises: a centre collection point (102) configured to receive the sample and
has a high abundance of the sample; and a circular blade (104) emerging from the
centre collection point and concentrically oriented around the centre collection
point. The blade distributes the sample into low abundance along its length.
[0055] In some embodiments, the present method comprises contacting an
antibody that specifically binds to the protein biomarkers and this may be
evaluated by western blotting or an assay. In a preferred embodiment, the assay
may be ELISA.
[0056] In an embodiment, the present disclosure provides a kit for detecting one
or more protein biomarkers associated with endometrial cancer, cervical cancer,
or fallopian tube cancer in a sample comprising: (a) a sample pad for receiving the
sample; (b) a conjugate area downstream of the sample pad comprising one or
more antibodies that bind specifically to the protein biomarkers to form
complexes; (c) one or more test line(s) downstream of the conjugate area
comprising labeled reagents for capturing the complexes; and (d) a control line
downstream of the test lines that indicates completion of assay.
[0057] In an embodiment, the kit may be used for point-of-care testing.
[0058] In an embodiment, the labeled reagents may include fluorescent labels,
radioactive labels, or enzymatic labels for detection of the complex.
[0059] In an embodiment, the kit is designed such that the sample flows through
the conjugate area, the test lines and finally reaches the control line. As the sample
flows through the kit, the protein biomarkers present in the sample bind to
corresponding anti-bodies in the conjugate area to form complexes. These

complexes then bind to the labeled reagents on the test line and indicate their presence via the labeling. In an embodiment, a single test line is specific to binding a single complex of a single protein biomarker.
[0060] While the foregoing describes various embodiments of the disclosure, other and further embodiments of the disclosure may be devised without departing from the basic scope thereof. The scope of the invention is determined by the claims that follow. The invention is not limited to the described embodiments, versions or examples, which are included to enable a person having ordinary skill in the art to make and use the invention when combined with information and knowledge available to the person having ordinary skill in the art.
ADVANTAGES OF THE PRESENT INVENTION
[0061] The present disclosure provides a method of screening gynecological
malignancies that is non-invasive.
[0062] The present disclosure provides a method of screening gynecological
malignancies that is simple and robust.
[0063] The present disclosure provides a method of screening gynecological
malignancies at an early stage which initiates an early treatment of cancer.

We Claim:

1. An in vitro method of screening one or more protein biomarkers associated with endometrial cancer, cervical cancer, or fallopian tube cancer in a sample, wherein the method comprises the steps of: (a) culturing the sample; (b) separating the protein biomarkers from the sample; (c) analyzing the protein biomarkers in the sample by mass-spectrometry to give a protein biomarker profile of the sample; and (d) comparing the protein biomarker profile with a reference protein biomarker profile; wherein the sample is menstrual blood or vaginal discharge.
2. The method as claimed in claim 1, wherein the protein biomarkers are separated from the sample by liquid chromatography.
3. The method as claimed in claim 1, wherein comparing the protein biomarker profile with reference protein biomarker profile is performed via computational analysis.
4. A device for collecting a sample for in vitro screening of endometrial cancer, cervical cancer, or fallopian tube cancer; wherein the device comprises: a centre collection point (102) configured to receive the sample and has a high abundance of the sample; and a circular blade (104) emerging from the centre collection point and concentrically oriented around the centre collection point and is configured to gradually distribute the sample away from the centre collection point; wherein the sample is menstrual blood or vaginal discharge.
5. The device as claimed in claim 4, wherein the circular blade has a gradual decrease in abundance of the sample along its length.
6. A kit for detecting one or more protein biomarkers associated with endometrial cancer, cervical cancer, or fallopian tube cancer in a sample comprising: (a) a

sample pad for receiving the sample; (b) a conjugate area downstream of the sample pad comprising one or more antibodies that bind specifically to the protein biomarkers to form complexes; (c) one or more test line(s) downstream of the conjugate area comprising labeled reagents for capturing the complexes; and (d) a control line downstream of the test lines that indicates completion of assay.