FORM 2
THE PATENTS ACT, 1970
(39 of 1970)
&
THE PATENTS RULES, 2003
COMPLETE SPECIFICATION
(See section 10, rule 13)
1. Title of the invention: METHOD FOR ANALYSING PLURIPOTENT STEM CELL
BIOMARKERS, AND IMPLEMENTATIONS THEREOF
2. Applicant(s)
NAME NATIONALITY ADDRESS
TZAR MANAGEMENT
TECHNOLOGY PRIVATE
LIMITED
Indian A-2303, Fair Field Lodha Luxuria,
Lodha Paradise, Near Majiwada
Bridge, Thane (West), Thane,
Maharashtra 400601, India
3. Preamble to the description
COMPLETE SPECIFICATION
The following specification particularly describes the invention and the manner in which it
is to be performed.
1
FIELD OF INVENTION
[001] The present disclosure broadly relates to the field of pluripotent stem cell
biomarkers, and particularly provides an in-vitro method for detecting and predicting
cancer by using the pluripotent stem cell biomarkers in a blood sample.
BACKGROUND 5 OF INVENTION
[002] Cancer is a major health concern worldwide, accounting for millions of deaths.
It has been reported that around 11 million people per year are diagnosed as patients
carrying tumours worldwide, and it is speculated that this number will increase to more
than 16 million by the year 2020 (Ferlay et al. International journal of cancer 136.5
10 E359-E386. 2015). The biologic heterogeneity of this disease and the vast populations
afflicted by it, pose the pivotal questions of when and whom to treat and with which
therapies. These important questions can only be addressed through the development
of more accurate and informative biomarkers.
[003] The treatment for cancer very much depends on the stage at which the disease
15 is diagnosed. With the technological improvements happening at a global level,
chances of treating cancer have been increased in the last few decades. The check point
is the stage at which the diseases are detected. There are certain occasions where the
cancer cannot be treated if it is detected at an advanced stage. The chances of a
successful treatment increase if the cancer is diagnosed at a very early stage. The
20 method of detection plays a vital role in this aspect and the use of specific biomarkers
is an active field of research.
[004] The non-specific nature of cancer symptoms makes diagnosis difficult. In
certain cases, the patient remains asymptotic. Therefore, early signs and symptoms of
cancer are often neglected by the patient which provides an opportunity for the cancer
25 to spread in the absence of any medical intervention. By the time the patient seeks
2
medical help, the cancer may be out of reach of available clinical treatments. Moreover,
unavailability of good biomarkers is another hindrance for cancer treatment.
[005] Cancer can be detected by several ways, including the medical imaging, tissue
biopsy, and liquid biopsy. Once a possible cancer is detected it is usually diagnosed by
microscopic examination of a tissue sample from a tissue 5 biopsy. Detecting and
diagnosing cancers early on is essential when it comes to treatment outcome and
survival, especially when it comes to highly malignant tumors. From several decades,
the only method for detecting cancer in humans has been to remove surgically a little
portion of the tumour and to examine it with a microscope to look for cancer cells in
10 the tissue. Unfortunately, this surgical intervention can only be carried out when the
tumour has attained a certain volume or begins to be the cause of functional disorders.
As this method can be used only when clinical symptoms are present, it cannot be
considered as the best one for an early diagnosis of cancer or, in other words, it cannot
be used as a screening test routinely made before any clinical sign of cancer. Therefore,
15 there is a need to develop a simple yet highly sensitive and specific cancer detection
systems and methods to overcome the above and other problems.
[006] Biomarkers are not only important for diagnostic purposes but can also be of
great prognostic value. With the identification of the right biomarker, the cancer
progression and effect and success of chemotherapeutic drugs can be evaluated in great
20 details. There is an urgent need to identify reliable biomarkers for different cancers and
also to develop methods for efficient detection and prediction of cancer and/or
metabolically altered and/or quiescent cells.
SUMMARY OF INVENTION
[007] In an aspect of the present disclosure, there is provided an in-vitro method for
25 detecting presence of metabolically altered cells, said method comprising: (a) obtaining
a blood sample; (b) enriching pluripotent stem cells from the blood sample, to obtain a
mixture comprising said pluripotent stem cells; (c) obtaining nucleic acid from the
3
mixture of step (b); (d) performing an assay with the nucleic acid for analysing
expression level of at least one biomarker of pluripotent stem cell; and (e) comparing
the expression level of the at least one biomarker of pluripotent stem cell in the sample
with an expression level of the at least one biomarker in a control sample, wherein an
increase in the expression level of the at least one biomarker in 5 the sample as compared
to the expression level of the at least one biomarker in the control sample detects the
presence of metabolically altered cells.
[008] In a second aspect of the present disclosure, there is provided an in-vitro method
for detecting presence of quiescent cells, said method comprising: (a) obtaining a blood
10 sample; (b) enriching pluripotent stem cells from the blood sample, to obtain a mixture
comprising said pluripotent stem cells; (c) obtaining nucleic acid from the mixture of
step (b); (d) performing an assay with the nucleic acid for analysing expression level
of at least one biomarker of pluripotent stem cell; and (e) comparing the expression
level of the at least one biomarker of pluripotent stem cell in the sample with an
15 expression level of the at least one biomarker in a control sample, wherein an increase
in the expression level of the at least one biomarker in the sample as compared to the
expression level of the at least one biomarker in the control sample detects the presence
of quiescent cells.
[009] In a third aspect of the present disclosure, there is provided an in-vitro method
20 for detecting cancer, said method comprising: (a) obtaining a blood sample; (b)
enriching pluripotent stem cells from the blood sample, to obtain a mixture comprising
said pluripotent stem cells; (c) obtaining nucleic acid from the mixture of step (b); (d)
performing an assay with the nucleic acid for analysing expression level of at least one
biomarker of pluripotent stem cell; and (e) comparing the expression level of the at
25 least one biomarker of pluripotent stem cell in the sample with an expression level of
the at least one biomarker in a control sample, wherein an increase in the expression
level of the at least one biomarker in the sample as compared to the expression level of
the at least one biomarker in the control sample detects cancer.
4
[0010] In a fourth aspect of the present disclosure, there is provided an in-vitro method
for predicting cancer, said method comprising: (a) obtaining a blood sample; (b)
enriching pluripotent stem cells from the blood sample, to obtain a mixture comprising
said pluripotent stem cells; (c) obtaining nucleic acid from the mixture of step (b); (d)
performing an assay with the nucleic acid for analysing expression 5 level of at least one
biomarker of pluripotent stem cell; and (e) comparing the expression level of the at
least one biomarker of pluripotent stem cell in the sample with an expression level of
the at least one biomarker in a control sample, wherein an increase in the expression
level of the at least one biomarker in the sample as compared to the expression level of
10 the at least one biomarker in the control sample predicts cancer.
[0011] In a fifth aspect of the present disclosure, there is provided an in-vitro method
for monitoring response to cancer treatment, said method comprising: (a) obtaining a
blood sample at a time point during an anti-cancer therapy; (b) enriching pluripotent
stem cells from the blood sample, to obtain a mixture comprising said pluripotent stem
15 cells; (c) obtaining nucleic acid from the mixture of step (b); (d) performing an assay
with the nucleic acid for analysing expression level of at least one biomarker of
pluripotent stem cell; and (e) comparing the expression level of the at least one
biomarker of pluripotent stem cell with an expression level of the at least one biomarker
of pluripotent stem cell in a reference sample to monitor the response to cancer
20 treatment.
[0012] In a sixth aspect of the present disclosure, there is provided an in-vitro method
for detecting a positive response to cancer treatment, said method comprising: (a)
obtaining a blood sample-I, before administration of an anti-cancer therapy; (b)
obtaining a blood sample-II, after administration of the anti-cancer therapy; (c)
25 enriching pluripotent stem cells from the blood sample-I to obtain a mixture-I
comprising said pluripotent stem cells; (d) enriching pluripotent stem cells from the
blood sample-II to obtain a mixture-II comprising said pluripotent stem cells; (e)
obtaining nucleic acid-I from the mixture-I; (f) obtaining nucleic acid-II from the
mixture-II; (g) independently performing an assay with the nucleic acid-I and the
5
nucleic acid-II for analysing expression level of at least one biomarker of pluripotent
stem cell; and (h) comparing the expression levels of the at least one biomarker of
pluripotent stem cell from the nucleic acid-II with the expression level of the at least
one biomarker of pluripotent stem cell from the nucleic acid-I, wherein a decrease in
the expression level of the at least one biomarker of pluripotent 5 stem cell from the
nucleic acid-II as compared to the expression level of the at least one biomarker of
pluripotent stem cell from the nucleic acid-I detects a positive response to the cancer
treatment.
[0013] In a seventh aspect of the present disclosure, there is provided an in-vitro
10 method for detecting cancer, said method comprising: (a) obtaining a blood sample; (b)
enriching pluripotent stem cells from the blood sample, to obtain a mixture comprising
said pluripotent stem cells; (c) obtaining nucleic acid from the mixture; (d) performing
an assay with the nucleic acid for analysing expression level of at least one biomarker
of pluripotent stem cell; (e) comparing the expression level of the at least one biomarker
15 of pluripotent stem cell in the sample with an expression level of the at least one
biomarker in a control sample, wherein an increase in the expression level of the at
least one biomarker in the sample as compared to the expression level of the at least
one biomarker in the control sample detects presence of cancer; (f) performing
sequence-based assays on the nucleic acid and analysing for mutation in at least one
20 cancer-related marker, wherein presence of mutation in the at least one cancer-related
marker indicates presence of a specific type of cancer based on the cancer-related
marker analysed.
[0014] In an eighth aspect of the present disclosure, there is provided a use of a
pluripotent stem cell biomarker selected from a group consisting of Oct-4, Sox-2,
25 Nanog, p53, NFκB, Sirt-1, Sirt-6, Sirt-3, NAD, RAS, ERC, erbB-2, ABL, subsets
thereof, and combinations thereof, for detecting cancer from a blood sample.
[0015] In a ninth aspect of the present disclosure, there is provided a use of a
pluripotent stem cell biomarker selected from a group consisting of Oct-4, Sox-2,
6
Nanog, p53, NFκB, Sirt-1, Sirt-6, Sirt-3, NAD, RAS, ERC, erbB-2, ABL, subsets
thereof, and combinations thereof, for predicting cancer from a blood sample.
[0016] In a tenth aspect of the present disclosure, there is provided a use of pluripotent
stem cell biomarker selected from a group consisting of Oct-4, Sox-2, Nanog, p53,
NFκB, Sirt-1, Sirt-6, Sirt-3, NAD, RAS, ERC, erbB-5 2, ABL, subsets thereof, and
combinations thereof, for grading stage of cancer from a blood sample.
[0017] In an eleventh aspect of the present disclosure, there is provided a use of a
pluripotent stem cell biomarker selected from a group consisting of Oct-4, Sox-2,
Nanog, p53, NFκB, Sirt-1, Sirt-6, Sirt-3, NAD, RAS, ERC, erbB-2, ABL, subsets
10 thereof, and combinations thereof, for monitoring progression of anti-cancer therapy
from a blood sample.
[0018] In a twelfth aspect of the present disclosure, there is provided a method for
treating cancer, said method comprising: (a) obtaining a blood sample from a subject;
(b) enriching pluripotent stem cells from the blood sample, to obtain a mixture
15 comprising said pluripotent stem cells; (c) obtaining nucleic acid from the mixture of
step (b); (d) performing an assay with the nucleic acid for analysing expression level
of at least one biomarker of pluripotent stem cell; (e) comparing the expression level
of the at least one biomarker of pluripotent stem cell in the sample with an expression
level of the at least one biomarker in a control sample, wherein an increase in the
20 expression level of the at least one biomarker in the sample as compared to the
expression level of the at least one biomarker in the control sample detects cancer; and
(f) administering an anti-cancer therapy to the subject for treating cancer.
[0019] These and other features, aspects, and advantages of the present subject matter
will be better understood with reference to the following description and appended
25 claims. This summary is provided to introduce a selection of concepts in a simplified
form. This summary is not intended to identify key features or essential features of the
claimed subject matter, nor is it intended to be used to limit the scope of the claimed
subject matter.
7
BRIEF DESCRIPTION OF ACCOMPANYING DRAWINGS
[0020] The following drawings form a 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 5 embodiments presented herein.
[0021] Figure 1 depicts the fold-change value for Sox 2, obtained with analysing
twenty different samples, in accordance with an embodiment of the present disclosure.
[0022] Figure 2 depicts the fold-change value for Nanog obtained with analysing
twenty different samples, in accordance with an embodiment of the present disclosure.
10 [0023] Figure 3 depicts the fold-change value for Oct-4a obtained with analysing
twenty different samples, in accordance with an embodiment of the present disclosure.
[0024] Figure 4 depicts the fold-change value for Sirt 1 obtained with analysing twenty
different samples, in accordance with an embodiment of the present disclosure.
[0025] Figure 5 depicts the fold-change value for Sirt 6 obtained with analysing twenty
15 different samples, in accordance with an embodiment of the present disclosure.
[0026] Figure 6 depicts the fold-change value for NFκB obtained with analysing
twenty different samples, in accordance with an embodiment of the present disclosure.
[0027] Figure 7 depicts the fold-change value for Oct-4 obtained with analysing twenty
different samples, in accordance with an embodiment of the present disclosure.
20 [0028] Figure 8 depicts the fold-change value for p53 obtained with analysing twenty
different samples, in accordance with an embodiment of the present disclosure.
[0029] Figure 9 depicts a representative first graph showing the fold-change value for
at least one biomarker of pluripotent stem cell as disclosed in the present study with a
cohort of 1000 samples, in accordance with an embodiment of the present disclosure.
25 The green tick marks below show concurrence with the actual clinical status by an
independent reviewer in the blinded study.
[0030] Figure 10 a representative second graph showing the fold-change value for at
least one biomarker of pluripotent stem cell as disclosed in the present study with a
8
cohort of 1000 samples, in accordance with an embodiment of the present disclosure.
The green tick marks below show concurrence with the actual clinical status by an
independent reviewer in the blinded study.
[0031] Figure 11 depicts a representative third graph showing the fold-change value
for at least one biomarker of pluripotent stem cell as disclosed 5 in the present study with
a cohort of 1000 samples study, in accordance with an embodiment of the present
disclosure. The green tick marks below show concurrence with the actual clinical status
by an independent reviewer in the blinded study.
[0032] Figure 12 depicts a representative fourth graph showing the fold-change value
10 for at least one biomarker of pluripotent stem cell as disclosed in the present study with
a cohort of 1000 samples study, in accordance with an embodiment of the present
disclosure. The green tick marks below show concurrence with the actual clinical status
by an independent reviewer in the blinded study.
[0033] Figure 13 depicts a representative fifth graph showing the fold-change value for
15 at least one biomarker of pluripotent stem cell as disclosed in the present study with a
cohort of 1000 samples, in accordance with an embodiment of the present disclosure.
The green tick marks below show concurrence with the actual clinical status by an
independent reviewer in the blinded study.
[0034] Figure 14 depicts a representative sixth graph showing the fold-change value
20 for at least one biomarker of pluripotent stem cell as disclosed in the present study with
a cohort of 1000 samples study, in accordance with an embodiment of the present
disclosure. The green tick marks below show concurrence with the actual clinical status
by an independent reviewer in the blinded study.
[0035] Figure 15 depicts a representative seventh graph showing the fold-change value
25 for at least one biomarker of pluripotent stem cell as disclosed in the present study with
a cohort of 1000 samples study, in accordance with an embodiment of the present
disclosure. The green tick marks below show concurrence with the actual clinical status
by an independent reviewer in the blinded study.
9
[0036] Figure 16 depicts a representative eighth graph showing the fold-change value
for at least one biomarker of pluripotent stem cell as disclosed in the present study with
a cohort of 1000 samples study, in accordance with an embodiment of the present
disclosure. The green tick marks below show concurrence with the actual clinical status
by an independent 5 reviewer in the blinded study.
[0037] Figure 17 depicts a representative ninth graph showing the fold-change value
of for least one biomarker of pluripotent stem cell as disclosed in the present study with
a cohort of 1000 samples study, in accordance with an embodiment of the present
disclosure. The green tick marks below show concurrence with the actual clinical status
10 by an independent reviewer in the blinded study.
[0038] Figure 18 depicts a representative tenth graph showing the fold-change value
for at least one biomarker of pluripotent stem cell as disclosed in the present study with
a cohort of 1000 samples study, in accordance with an embodiment of the present
disclosure. The green tick marks below show concurrence with the actual clinical status
15 by an independent reviewer in the blinded study.
DETAILED DESCRIPTION OF THE INVENTION
[0039] Those skilled in the art will be aware that the present disclosure is subject to
variations and modifications other than those specifically described. It is to be
understood that the present disclosure includes all such variations and modifications.
20 The disclosure also includes all such steps, features, compositions, and compounds
referred to or indicated in this specification, individually or collectively, and any and
all combinations of any or more of such steps or features.
Definitions
[0040] For convenience, before further description of the present disclosure, certain
25 terms employed in the specification, and examples are delineated here. These
definitions should be read in the light of the remainder of the disclosure and understood
as by a person of skill in the art. The terms used herein have the meanings recognized
10
and known to those of skill in the art, however, for convenience and completeness,
particular terms and their meanings are set forth below.
[0041] The articles “a”, “an” and “the” are used to refer to one or to more than one
(i.e., to at least one) of the grammatical object of the article.
[0042] The terms “comprise” and “comprising” 5 are used in the inclusive, open sense,
meaning that additional elements may be included. It is not intended to be construed as
“consists of only”.
[0043] Throughout this specification, unless the context requires otherwise the word
“comprise”, and variations such as “comprises” and “comprising”, will be understood
10 to imply the inclusion of a stated element or step or group of element or steps but not
the exclusion of any other element or step or group of element or steps.
[0044] The term “including” is used to mean “including but not limited to”.
“Including” and “including but not limited to” are used interchangeably.
[0045] Ratios, concentrations, amounts, and other numerical data may be presented
15 herein in a range format. It is to be understood that such range format is used merely
for convenience and brevity and should be interpreted flexibly to include not only the
numerical values explicitly recited as the limits of the range, but also to include all the
individual numerical values or sub-ranges encompassed within that range as if each
numerical value and sub-range is explicitly recited For example, a ratio of about 1:1 to
20 1:20 should be interpreted to include not only the explicitly recited limits of about 1:1
to about 1:20, but also to include sub-ranges, such as 1:2 to 1:10, 1:2 to 1:15, and so
forth, as well as individual amounts, including fractional amounts, within the specified
ranges, such as 1:2.5, and 1:16.3, for example.
[0046] The term “cancer” refers to the physiological condition in animals that is
25 characterized by unregulated cell growth. The term “cancer” as used in the present
disclosure is intended to include benign and malignant cancers, dormant tumours or
micro-metastases. The types of cancer include, but are not limited to, carcinoma,
lymphoma, blastoma (including medulloblastoma and retinoblastoma), sarcoma
(including liposarcoma and synovial cell sarcoma), neuroendocrine tumours (including
11
carcinoid tumours, gastrinoma, and islet cell cancer), mesothelioma, schwannoma
(including acoustic neuroma), meningioma, adenocarcinoma, melanoma, and
leukaemia or lymphoid malignancies. More particular examples of cancers include
breast cancer, liver cancer, ovarian cancer, lung cancer, leukaemia, prostate cancer,
lymphoma, pancreatic cancer, cervical cancer, colon cancer, 5 osteosarcoma, testicular
cancer, thyroid cancer, gastric cancer, Ewing sarcoma, bladder cancer, gastrointestinal
stromal tumour (GIST), kidney cancer (e.g., renal cell carcinoma), squamous cell
cancer (e.g., epithelial squamous cell cancer), lung cancer (including small - cell lung
cancer (SCLC), non-small cell lung cancer (NSCLC), adenocarcinoma of the lung,
10 squamous carcinoma of the lung, cancer of the peritoneum, hepatocellular cancer,
gastric or stomach cancer including gastrointestinal cancer, pancreatic cancer,
glioblastoma, cervical cancer, ovarian cancer, liver cancer, hepatoma, breast cancer
(including metastatic breast cancer), bladder cancer, colon cancer, rectal cancer,
colorectal cancer, endometrial or uterine carcinoma, salivary gland carcinoma, prostate
15 cancer, vulval cancer, thyroid cancer, hepatic carcinoma, anal carcinoma, penile
carcinoma, Merkel cell cancer, mycoses fungoids, testicular cancer, oesophageal
cancer, tumours of the biliary tract, head and neck cancer, as well as B-cell lymphoma
(including low grade/follicular non-Hodgkin's lymphoma (NHL); small lymphocytic
(SL) NHL; intermediate grade / follicular NHL; inter mediate grade diffuse NHL; high
20 grade immunoblastic NHL; high grade lymphoblastic NHL; high grade small non -
cleaved cell NHL; bulky disease NHL, mantle cell lymphoma; AIDS-related
lymphoma; and Waldenstrom's Macroglobulinemia; chronic lymphocytic leukaemia
(CLL); acute lymphoblastic leukaemia (ALL); Hairy cell leukaemia; chronic
myeloblastic leukaemia; and post-transplant lymphoproliferative disorder (PTLD), as
25 well as abnormal vascular proliferation associated with phakomatoses, edema (such as
that associated with brain tumours), and Meigs’ syndrome.
[0047] The term “advanced stage of cancer” or “cancer at advanced stage” refers to a
cancer which has spread outside the site of the organ of origin, either by local origin or
by metastasis. The terms “stage-I”, “stage-II”, “stage-III”, and “stage-IV” are well-
12
known terms used to refer to a grade of cancer which has inflicted a patient. The term
“pre-cancerous” is used to refer to a stage of cancer where no symptoms of the cancer
is visible. The pre-cancerous stage could not be detected by a PET scan. The term
“early detection” as it relates to cancer, has been used to describe detection of stage-I,
or stage-II cancer. The term “early onset” refers to a cancer 5 at a stage where it is not
yet detectable by any one of the conventional methods known in the art. The term
“detects” or “detection” refers to a detection which has been performed outside of a
patient/subject using a sample from the patient/subject.
[0048] The term “predicts”, or “prediction” refers to a likelihood of something that will
10 happen in future or in due course of time.
[0049] The term “blood sample” refers to the whole blood sample that is obtained from
a subject. The scope of the method as disclosed herein begins from the stage of having
obtained the blood sample irrespective of the source for the procurement, and the
method does not involve any invasive techniques or operating on a subject. The term
15 “blood sample” encompasses any form of processed blood sample also. By
“processing”, the present disclosure intends to cover any method for enriching a
specific population of cells or a mere processing so as to enable the blood sample to be
used for testing by “in-vitro” methods.
[0050] The term “in-vitro” refers to a task or method or experiment being performed
20 or taking place in a test tube, culture dish, or elsewhere outside a living organism.
[0051] The term “reference” refers to at least one selected from a group consisting of:
(a) blood sample obtained prior to administering anti-cancer therapy; (b) blood sample
obtained prior to the time point at which a blood sample under study is obtained; (c)
blood sample obtained at a time point subsequent to which the blood sample under
25 study is obtained; and (d) blood sample obtained from a control. The term “reference
level” refers to the expression of at least one biomarker of pluripotent stem cell
obtained from the reference. The term “control sample” refers to a blood sample
obtained from a non-cancer subject, and the term “expression of at least one biomarker
of pluripotent stem cell in a control sample” refers to the expression of the at least one
13
biomarker of pluripotent stem cell as disclosed herein, and the expression is studied
using similar steps as is disclosed herein for studying the expression of the biomarker
in the blood sample. It is understood that the steps for processing and analysing
expression of the biomarker in a control sample is similar to the steps for performing
and analysing expression of the biomarker in a blood sample, 5 and the expression levels
of the biomarker obtained from the blood sample in compared with the expression
levels obtained from the control sample.
[0052] The term “expression level” refers to a particular level of expression of a
nucleic acid. The nucleic acid can be a DNA or RNA. The DNA is intended to include
10 cDNA, and RNA is intended to include all types of RNA, including mRNA. The term
“increase in the expression level” refers to an increased expression or increased levels
of a biomarker in an individual relative to a control, such as an individual or individuals
who are not suffering from the disease or disorder (e.g., cancer), an internal control
(e.g., a housekeeping biomarker), or the level of a biomarker in a sample obtained prior
15 to administration of a therapy.
[0053] The term “metabolically altered cell” refers to any cell which is altered
metabolically to a form that is not supposed to be present in an environment under
normal circumstances. The alteration can be in form of an increase in proliferation.
[0054] The term “quiescent cell” refers to a quiescent cell that does not proliferate as
20 per the regulated cell cycle of division.
[0055] The term “pluripotent stem cell” refers to cell which has the ability of selfreplicating
and to give rise to all types of cells in a subject.
[0056] The term “biomarker” as used herein refers to a biomolecule which is a nucleic
acid and is used to characterize a particular cell population. The term is intended to
25 cover both DNA and RNA forms of nucleic acid. The term “biomarker of pluripotent
stem cell” refers to any biomarker which can be used to characterize a population of
pluripotent stem cells.
14
[0057] The term “subject” refers to any mammal whose blood sample has been taken
for analysis using the in-vitro method of the present disclosure. The exemplification is
based on human beings used as subjects.
[0058] The term “cancer-free” refers to a subject who has not been diagnosed with
cancer. The term “positive response” as used herein refers 5 to a positive response of a
subject to anti-cancer therapy which means the therapy is effective in reducing the
population of cancerous cells. The term “negative response” as used herein refers that
the anti-cancer therapy is not able to reduce the population of cancerous cells or not
able to cure the subject of cancer.
10 [0059] The term “invasive” refers to any technique that involves entry into the living
body as by way of incision or by way of insertion of an instrument.
[0060] The term “at least one biomarker of pluripotent stem cell” refers to a gene which
is selected from a group consisting of: Oct-4 (octamer-binding transcription factor 4),
Sox-2 (sex determining region Y-box 2), Nanog, p53, NFκB, Sirt-1 (Sirtuin 1), Sirt-6
15 (Sirtuin 6), NAD (Nicotinamide adenine dinucleotide), RAS, ERC, erbB-2 (Erb-B2
Receptor Tyrosine Kinase 2), ABL (Abelson murine leukemia), subsets thereof, and
combinations thereof. The subsets of Oct-4, Sox-2, Nanog, p53, NFκB, Sirt-1, Sirt-6,
NAD, RAS, ERC, erbB-2, ABL and combinations thereof are also part of the present
disclosure.
20 [0061] The term “cancer-related marker” comprises all the well-known cancer-related
marker in the field of cancer study. A non-limiting list of cancer-related markers is
mentioned herewith and includes, ABL1, EVI1, MYC, APC, IL2, TNFAIP3, ABL2,
EWSR1, MYCL1, ARHGEF12, JAK2, TP53, AKT1, FEV, MYCN, ATM, MAP2K4,
TSC1, AKT2, FGFR1, NCOA4, BCL11B, MDM4, TSC2, ATF1, FGFR1OP, NFΚB2,
25 BLM, MEN1, VHL, BCL11A, FGFR2, NRAS, BMPR1A, MLH1, WRN, BCL2, FUS,
NTRK1, BRCA1, MSH2, WT1, BCL3, GOLGA5, NUP214, BRCA2, NF1, BCL6,
GOPC, PAX8, CARS, NF2, BCR, HMGA1, PDGFB, CBFA2T3, NOTCH1, BRAF,
HMGA2, PIK3CA, CDH1, NPM1, CARD11, HRAS, PIM1, CDH11, NR4A3, CBLB,
IRF4, PLAG1, CDK6, NUP98, CBLC, JUN, PPARG, CDKN2C, PALB2, CCND1,
15
KIT, PTPN11, CEBPA, PML, CCND2, KRAS, RAF1, CHEK2, PTEN, CCND3, LCK,
REL, CREB1, RB1, CDX2, LMO2, RET, CREBBP, RUNX1, CTNNB1, MAF, ROS1,
CYLD, SDHB, DDB2, MAFB, SMO, DDX5, SDHD, DDIT3, MAML2, SS18, EXT1,
SMARCA4, DDX6, MDM2, TCL1A, EXT2, SMARCB1, DEK, MET, TET2,
FBXW7, SOCS1, EGFR, MITF, TFG, FH, STK11, ELK4, 5 MLL, TLX1, FLT3, SUFU,
ERBB2, MPL, TPR, FOXP1, SUZ12, ETV4, MYB, USP6, GPC3, SYK, ETV6, IDH1,
TCF3, and combinations thereof. The list provided herein refers to the list of cancerrelated
markers which are well-known and common to a skilled person in the art. The
abbreviated forms are construed to be known to skilled person in the art.
10 [0062] The term “administering” refers to a method of giving a dosage of a compound
(e.g., a VEGF antagonist and / or a PD - L1 axis binding antagonist) or a composition
(a pharmaceutical composition including a VEGF antagonist and / or a PD - L1 axis
binding antagonist) to a patient. The administration can be intramuscular, intravenous,
intradermal, percutaneous, intraarterial, intraperitoneal, intralesional, intracranial,
15 intraarticular, intraprostatical, intrapleural, intratracheal, intrathecal, intranasal,
intravaginal, intrarectal, topical, intratumoral, peritoneal, subcutaneous,
subconjunctival, intravesicular, mucosal, intrapericardial, intraumbilical, intraocular,
intraorbital, intravitreal, oral.
[0063] The term “anti-cancer therapy” refers to any therapy known in the art for
20 curing/treating cancer.
[0064] The term “chemotherapeutic agent” refers to chemical compounds useful in the
treatment of cancer.
[0065] The term “enriching” as per the present disclosure refers to a process for
isolating a required population of cell in a manner that the required population is
25 present at a higher population in the isolated mixture which would be beneficial to
analyse a biomarker in such population. The present disclosure mentions “enriching”
in a context where it implies to mention a process for increasing concentration of a
particular type of pluripotent stem cells so as to enable studies to be carried out for
analysing expression level of at least one biomarker of pluripotent stem cell and/or for
16
analysing mutation in nucleic acid obtained from such population of pluripotent stem
cell.
[0066] Unless defined otherwise, all technical and scientific terms used herein have
the same meaning as commonly understood by one of ordinary skill in the art to which
this disclosure belongs. Although any methods and materials 5 similar or equivalent to
those described herein can be used in the practice or testing of the disclosure, the
preferred methods, and materials are now described. All publications mentioned herein
are incorporated herein by reference.
[0067] The methods currently available for detection of cancer have several drawbacks
10 associated with them. The limitations associated with Positron emission tomography
(PET) and circulating tumour cells (CTC) have been described below.
Limitations of PET scan
[0068] Positron emission tomography (PET), now almost 45 years after its initial
development, has become an established nuclear imaging modality that has proved
15 especially useful in cancer diagnosis. PET makes use of a tracer molecular known as
18F-2-fluoro-2-deoxy-D-glucose (FDG) (an analogue of glucose). The use of FDG to
image glucose metabolic rate takes advantage of the observation, that malignant cells
have higher rates of aerobic glycolysis than normal tissues (Griffeth 2005, BUMC
Proceedings, 18:321–330). Thus, the malignant cell utilizes more glucose to meet their
20 energy needs. FDG is currently the only agent approved by the Food and Drug
Administration (FDA) for oncology studies. Fortunately, while FDG is not a perfect
imaging agent (some tumours show poor FDG avidity and some benign processes show
high FDG avidity), FDG does work very well in most malignant tumours of clinical
importance, with the largest exception being prostate cancer.
25 [0069] Several factors can make the interpretation of PET studies challenging. Chief
among these factors in daily practice are variable physiologic uptake of FDG by normal
tissues, FDG uptake related to inflammation or infection, occasional malignant lesions
17
with low avidity for FDG, unusual tumour sites, limited resolution of small lesions,
altered biodistribution of FDG related to hyperglycemia or hyperinsulinemia, bone
marrow activation commonly encountered in cancer patients, and motion artefacts.
Unfortunately, glucose uptake is prevalent in cells of the body other than malignant
cells. Physiologic uptake in some normal tissues can 5 be highly variable. Although
many accumulate FDG to a predictable extent, there are others whose uptake cannot be
predicted. For example, the brain typically shows intense uptake of FDG, because it
metabolizes glucose exclusively, while myocardial uptake is intense in patients who
have not fasted but highly variable in patients who have fasted. Adipose tissue typically
10 shows minimal FDG uptake, but certain adipose deposits (so-called “brown fat”) that
play a role in thermogenesis can be dramatically activated in a cold or nervous patient.
Sometimes, even relatively predictable activity can be confusing. For example, unlike
glucose, FDG is not well reabsorbed by the proximal tubules of the kidney. Thus, it
can be predicted that intense activity will be seen in the kidneys and bladder. However,
15 focal pooling of excreted activity in a ureter could be confused with a hypermetabolic
iliac lymph node metastasis. Inflammatory cells, especially macrophages, can
sometimes accumulate FDG to a considerable extent, so inflammatory or infectious
sites are sometimes visualized on PET. Granulomatous conditions, such as sarcoidosis,
fungal infections, tuberculosis, and Mycobacterium avium-intracellular infection, can
20 cause particular problems in the PET evaluation of pulmonary lesions or lymph nodes.
Even inflammation related to therapeutic procedures, such as surgery or radiotherapy,
can cause significant uptake. When clinically possible, it is usually wise to wait at least
3 months after the completion of radiotherapy before performing a PET study to avoid
confusion by inflammatory uptake of FDG. Therefore, it is not possible to perform PET
25 within short span of intervals for checking the response of a patient undergoing cancer
treatment, one has to wait for a minimum time period of 3 months to check whether the
patient is responding to the treatment. If the patient is not responding, then losing the
precious time shall prove to be fatal to the patient.
18
[0070] Some malignant lesions have low avidity for FDG such as prostate,
bronchoalveolar cell carcinoma, low-grade sarcomas, certain low-grade non-
Hodgkin’s lymphomas, and even a few well-differentiated adenocarcinomas of the
lung—that may show poor concentration of FDG. Most neuroendocrine tumours are
poorly seen on FDG-PET. Small lesions or unusual presentations/5 locations always
make the job of tumour detection and staging more difficult. PET alone is sometimes
unable to localize small tumours or confirm whether FDG uptake in unusual sites
reflects tumour or nontumor.
Limitations of Circulating Tumour Cells
10 [0071] Despite the numerous scientific publications related to CTC detection in cancer
patients, the physician does not use this biomarker in routine clinical practice. This can
be explained by the large number of methods available for CTC detection and by the
difficulty of the physician and the biologist to select the optimal method (Huang T et
al, Biosens Bioelectron 2014; 51:213-8). In this context, it is noteworthy that the only
15 FDA approved method for CTC detection, the CellSearch method (Janssen Diagnostics
Company, USA), has been approved for CTC detection in metastatic breast, prostate
and colon cancer patients. Contrarily, CTC have been reported to result in false
positives and false negative in the detection for breast, prostate and colon cancer (Lori
M. Millner et al, Ann Clin Lab Sci. 2013 43(3), because CTCs that have undergone
20 epithelial-mesenchymal transition cannot express epithelial biomarkers, the CellSearch
system can certainly miss the detection of a subpopulation of CTCs of interest in
several cancer patients (Hofman VJ et al, Am J Clin Pathol 2011;135). Direct
technologies for CTC detection in cancer are certainly strongly attractive, but the
results obtained by different teams probably need to be validated in independent and
25 large multi-centre studies. Many other methods are currently being developed for CTC
characterization, such as a method allowing functional evaluation the CTCs and
characterization of a subpopulation of malignant cells (Yao X et al, Integr Biol
19
(Camb) 2014;6:388-98). Currently these new methods seem to be difficult for
translation into the clinical routine practice. These approaches lack a multi-centre
assessment program, and thus it is difficult to evaluate their reproducibility, their
sensitivity and their specificity.
[0072] The existing technique for detecting cancer is based on 5 the specific cancer
biomarker isolated from the affected tissue, the method therefore involves invasive
methods to isolate the affected tissue for detecting the presence of cancer. The
techniques available at present offers detection of cancer which involves invasive
technique and for obvious reasons the technique cannot be done frequently on a patient
10 to check for the presence of cancer. Therefore, the existing techniques have drawbacks
in terms of difficulty in practising, not able to accurately predict the possibility of
cancer with a single practice on the patient, not fit for follow-up cure of the patient.
The existing technique can only be used to detect certain type of cancers once they
have reached an advanced stage, and the patient having reached an advance stage of
15 the disease has very less chance of surviving the treatment, this is one of the greatest
drawbacks of the existing technique.
[0073] There exist lacunae in terms of the availability of a simple test which can be
performed to detect all types of cancer at an early stage, and which can be performed
in a frequent manner to monitor the stage of cancer. The present disclosure discloses a
20 process for detecting and predicting cancer by analysing blood sample of the subject
concerned. The present disclosure discloses a process for detecting the grade of cancer
that a subject is suffering from, with a simple blood sample. The present disclosure
accurately detects the type of cancer with a simple blood sample, thereby without using
any invasive techniques such as biopsy. The process of the present disclosure predicts
25 the possibility of a subject getting inflicted with cancer with only a blood sample. The
present disclosure detects pre-cancerous stage even before any symptoms start to
appear, thus providing a significant advantage over conventional methods of detection
which can only detect cancer once it has reached a certain level in the body. The present
disclosure also discloses a process for enriching pluripotent stem cells (PSC) which
20
can further be used for evaluating stem cell markers on PSC. Thus, the present
disclosure discloses a simple, efficient, and highly accurate process for detecting and
predicting cancer by analysing a simple blood sample from a subject.
[0074] The present disclosure discloses in-vitro methods for detecting presence of
cancer, predicting the chances of getting inflicted with cancer, 5 grading stage of cancer,
monitoring cancer progression, monitoring response to anti-cancer treatment, followup
check to confirm whether cancer has been eradicated from the subject concerned.
The in-vitro method as disclosed herein also provides accurate information about
specific type of cancer with only a blood sample and not involving any invasive
10 techniques. The method as disclosed herein provides the information regarding type of
cancer much before the manifestation of conditions which are detectable by known
techniques such as PET scan, thereby, detecting the presence of specific type of cancer
without having the need to perform biopsy. Also, to enable a doctor to target a specific
tissue of organ for performing biopsy, the patient needs to show particular symptoms,
15 but in case of cancer the symptoms may appear at an advanced stage thereby reducing
the chances of patient’s survival, the in-vitro method of present disclosure not only
detects the presence of cancer but also detects the primary site of cancer and its type
much before the manifestation of symptoms with only a blood sample. The method as
disclosed in the present disclosure poses all the above-mentioned uses working only
20 with a blood sample. The simplicity of the method poses the benefit of being able to
use this method frequently while treating a patient for cancer, as against the use of PET
scan which can be done with a minimum interval of 6 months. The methods as
disclosed herein are in-vitro methods and free of any invasive techniques. The method
as disclosed in the present disclosure can detect/predict any cancer in a subject by
25 analysing the blood sample of the subject, said method comprising: (a) obtaining a
blood sample; (b) enriching pluripotent stem cells from the blood sample, to obtain a
mixture comprising said pluripotent stem cells; (c) obtaining nucleic acid from the
mixture of step (b); (d) performing an assay with the nucleic acid for analysing
expression level of at least one biomarker of pluripotent stem cell; and (e) comparing
21
the expression level of the at least one biomarker of pluripotent stem cell with a control,
wherein an increase in the expression level of the at least one biomarker as compared
to the control detects/predicts cancer. The method as disclosed in the present disclosure
is capable of detecting presence of metabolically altered cells/quiescent cells by
analysing a blood sample by a method comprising: (a) obtaining 5 a blood sample; (b)
enriching pluripotent stem cells from the blood sample, to obtain a mixture comprising
said pluripotent stem cells; (c) obtaining nucleic acid from the mixture of step (b); (d)
performing an assay with the nucleic acid for analysing expression level of at least one
biomarker of pluripotent stem cell; and (e) comparing the expression level of the at
10 least one biomarker of pluripotent stem cell with a control, wherein an increase in the
expression level of the at least one biomarker as compared to the control detects the
presence of metabolically altered cells. The method as disclosed in the present
disclosure is capable of monitoring response to cancer treatment by analysing a blood
sample, said method comprises: (a) obtaining a blood sample at one time point
15 following an anti-cancer therapy; (b) enriching pluripotent stem cells from the blood
sample to obtain a mixture comprising said pluripotent stem cells; (c) obtaining nucleic
acid from the mixture; (d) performing an assay with the nucleic acid for analysing
expression level of at least one biomarker of pluripotent stem cell; and (e) comparing
the expression level of the at least one biomarker of pluripotent stem cell with an
20 expression level of the at least one biomarker of pluripotent stem cell in a reference
that monitors the response to cancer treatment.
[0075] The present disclosure discloses a method for detecting presence of cancer and
also for detecting a specific type of cancer from a blood sample, said method
comprises: (a) obtaining a blood sample; (b) enriching pluripotent stem cells from the
25 blood sample, to obtain a mixture comprising said pluripotent stem cells; (c) obtaining
nucleic acid from the mixture; (d) performing an assay with the nucleic acid for
analysing expression level of at least one biomarker of pluripotent stem cell; (e)
comparing the expression level of the at least one biomarker of pluripotent stem cell
with a control, wherein an increase in the expression level of the at least one biomarker
22
as compared to the control indicates presence of cancer; (f) performing sequence-based
assays on the nucleic acid and analysing for mutation in at least one cancer-related
marker, wherein presence of mutation in the at least one cancer-related marker
indicates presence of a specific type of cancer based on the cancer-related marker
5 analysed.
[0076] The method as disclosed in the present disclosure is used for detecting and
predicting pre-cancerous stage, stage-I cancer, stage-II cancer, stage-III cancer, and
stage IV cancer, wherein the cancer is selected from a non-limiting group consisting of
ovarian cancer, breast cancer, prostate cancer, lung cancer, liver cancer, colon cancer,
10 leukaemia, lymphoma, bladder cancer, renal cancer, thyroid cancer, pancreatic cancer.
It is contemplated that other varieties of cancer types can also be included in the present
disclosure.
[0077] The present disclosure is not to be limited in scope by the specific embodiments
described herein, which are intended for the purposes of exemplification only.
15 Functionally-equivalent products, compositions, and methods are clearly within the
scope of the disclosure, as described herein.
[0078] Although the subject matter has been described with reference to specific
embodiments, this description is not meant to be construed in a limiting sense. Various
modifications of the disclosed embodiments, as well as alternate embodiments of the
20 subject matter, will become apparent to persons skilled in the art upon reference to the
description of the subject matter. It is therefore contemplated that such modifications
can be made without departing from the spirit or scope of the present subject matter as
defined.
[0079] In an embodiment of the present disclosure, there is provided an an in-vitro
25 method for detecting presence of metabolically altered cells, said method comprising:
(a) obtaining a blood sample; (b) enriching pluripotent stem cells from the blood
sample, to obtain a mixture comprising said pluripotent stem cells; (c) obtaining nucleic
acid from the mixture of step (b); (d) performing an assay with the nucleic acid for
analysing expression level of at least one biomarker of pluripotent stem cell; and (e)
23
comparing the expression level of the at least one biomarker of pluripotent stem cell in
the sample with an expression level of the at least one biomarker in a control sample,
wherein an increase in the expression level of the at least one biomarker in the sample
as compared to the expression level of the at least one biomarker in the control sample
detects the presence of metabolically 5 altered cells.
[0080] In an embodiment of the present disclosure, there is provided an an in-vitro
method for detecting presence of metabolically altered cells, said method comprising:
(a) obtaining a blood sample; (b) enriching pluripotent stem cells from the blood
sample, to obtain a mixture comprising said pluripotent stem cells; (c) obtaining nucleic
10 acid from the mixture of step (b); (d) performing an assay with the nucleic acid for
analysing expression level of at least one biomarker of pluripotent stem cell; and (e)
comparing the expression level of the at least one biomarker of pluripotent stem cell in
the sample with an expression level of the at least one biomarker in a control sample,
wherein an increase in the expression level of the at least one biomarker in the sample
15 as compared to the expression level of the at least one biomarker in the control sample
detects the presence of metabolically altered cells, and wherein the method further
comprises analysing the nucleic acid by performing sequence-based assays.
[0081] In an embodiment of the present disclosure, there is provided an an in-vitro
method for detecting presence of metabolically altered cells as described herein,
20 wherein the increase in the expression level of the at least one biomarker of pluripotent
stem cell is at least 2 folds as compared to the control. In another embodiment, the
increase in the expression level of the at least one biomarker of pluripotent stem cell is
at least 3 folds as compared to the control. In yet another embodiment, the increase in
the expression level of the at least one biomarker of pluripotent stem cell is at least 5
25 folds as compared to the control.
[0082] In an embodiment of the present disclosure, there is provided an an in-vitro
method for detecting presence of metabolically altered cells as described herein,
wherein the increase in the expression level of the at least one biomarker of pluripotent
stem cell is in a range of 10-20 folds as compared to the control. In another
24
embodiment, the increase in the expression level of the at least one biomarker of
pluripotent stem cell is in a range of 20-30 folds as compared to the control. In yet
another embodiment, the increase in the expression level of the at least one biomarker
of pluripotent stem cell is in a range of 30-40 folds as compared to the control. In one
another embodiment, the increase in the expression level of the 5 at least one biomarker
of pluripotent stem cell is in a range of 40-50 folds as compared to the control.
[0083] In an embodiment of the present disclosure, there is provided an an in-vitro
method for detecting presence of metabolically altered cells as described herein,
wherein the at least one biomarker of pluripotent stem cell is selected from a group
10 consisting of Oct-4, Sox-2, Nanog, p53, Sirt-1, Sirt-6, Sirt-3, NAD, RAS, ERC, erbB-
2, ABL, subsets thereof, and combinations thereof. In another embodiment, the at least
one biomarker of pluripotent stem cell is Oct-4. In yet another embodiment, the at least
one biomarker of pluripotent stem cell is Oct-4a. In an alternate embodiment, the at
least one biomarker of pluripotent stem cell is Oct-4b.
15 [0084] In an embodiment of the present disclosure, there is provided an an in-vitro
method for detecting presence of metabolically altered cells as described herein,
wherein the at least one biomarker of pluripotent stem cell is selected from a group
consisting of Oct-4, Sox-2, Nanog, p53, Sirt-1, Sirt-6, Sirt-3, subsets thereof, and
combinations thereof. In another embodiment, the at least one biomarker of pluripotent
20 stem cell is Sox-2. In yet another embodiment, the at least one biomarker of pluripotent
stem cell is Nanog. In an alternate embodiment, the at least one biomarker of
pluripotent stem cell is p53. In one another embodiment, the at least one biomarker of
pluripotent stem cell is Sirt-1. In another alternate embodiment, the at least one
biomarker of pluripotent stem cell is Sirt-6. In yet another alternate embodiment, the at
25 least one biomarker of pluripotent stem cell is Sirt-3.
[0085] In an embodiment of the present disclosure, there is provided an an in-vitro
method for detecting presence of metabolically altered cells as described herein,
wherein obtaining the nucleic acid from the mixture is by any one method selected
from a group consisting of: (a) guanidinium thiocyanate-phenol-chloroform nucleic
25
acid extraction; (b) cesium chloride gradient centrifugation method; (c)
cetyltrimethylammonium bromide nucleic acid extraction; (d) alkaline extraction; (e)
resin-based extraction; and (f) solid phase nucleic acid extraction.
[0086] In an embodiment of the present disclosure, there is provided an an in-vitro
method for detecting presence of metabolically altered cells 5 as described herein,
wherein performing an assay with the nucleic acid for analysing the expression of the
at least one biomarker is done by a technique selected from a group consisting of:
quantitative PCR, flow cytometry, and Next Generation Sequencing (NGS).
[0087] In an embodiment of the present disclosure, there is provided an an in-vitro
10 method for detecting presence of metabolically altered cells as described herein,
wherein the control is the expression level of the at least one biomarker of pluripotent
stem cells obtained from a cancer-free subject.
[0088] In an embodiment of the present disclosure, there is provided an an in-vitro
method for detecting presence of metabolically altered cells as described herein,
15 wherein the enriching of the pluripotent stem cells from the blood sample comprises:
(a) contacting the blood sample with a neutral buffer in a ratio range of 1:1 to 1:20, to
obtain a first mixture; (b) contacting at least one salt solution to the first mixture in a
ratio range of 1:2 to 1:10, to obtain a second mixture; and (c) processing the second
mixture to obtain enriched pluripotent stem cells.
20 [0089] In an embodiment of the present disclosure, there is provided an an in-vitro
method for detecting presence of metabolically altered cells as described herein,
wherein the enriching of the pluripotent stem cells from the blood sample comprises:
(a) contacting the blood sample with a neutral buffer in a ratio range of 1:1 to 1:20, to
obtain a first mixture; (b) contacting at least one salt solution to the first mixture in a
25 ratio range of 1:2 to 1:10, to obtain a second mixture; and (c) processing the second
mixture to obtain enriched pluripotent stem cells, and wherein the processing of the
second mixture comprises, at least one method selected from a group consisting of: (a)
extraction process; (b) washing process; (c) centrifugation process, and combinations
thereof.
26
[0090] In an embodiment of the present disclosure, there is provided an an in-vitro
method for detecting presence of metabolically altered cells as described herein,
wherein the method is independent of invasive techniques.
[0091] In an embodiment of the present disclosure, there is provided an an in-vitro
method for detecting presence of metabolically altered cells 5 as described herein,
wherein the nucleic acid is DNA. In another embodiment, the nucleic acid is RNA.
[0092] In an embodiment of the present disclosure, there is provided an in-vitro method
for detecting presence of quiescent cells, said method comprising: (a) obtaining a blood
sample; (b) enriching pluripotent stem cells from the blood sample, to obtain a mixture
10 comprising said pluripotent stem cells; (c) obtaining nucleic acid from the mixture of
step (b); (d) performing an assay with the nucleic acid for analysing expression level
of at least one biomarker of pluripotent stem cell; and (e) comparing the expression
level of the at least one biomarker of pluripotent stem cell in the sample with an
expression level of the at least one biomarker in a control sample, wherein an increase
15 in the expression level of the at least one biomarker in the sample as compared to the
expression level of the at least one biomarker in the control sample detects the presence
of quiescent cells.
[0093] In an embodiment of the present disclosure, there is provided an in-vitro method
for detecting presence of quiescent cells, said method comprising: (a) obtaining a blood
20 sample; (b) enriching pluripotent stem cells from the blood sample, to obtain a mixture
comprising said pluripotent stem cells; (c) obtaining nucleic acid from the mixture of
step (b); (d) performing an assay with the nucleic acid for analysing expression level
of at least one biomarker of pluripotent stem cell; and (e) comparing the expression
level of the at least one biomarker of pluripotent stem cell in the sample with an
25 expression level of the at least one biomarker in a control sample, wherein an increase
in the expression level of the at least one biomarker in the sample as compared to the
expression level of the at least one biomarker in the control sample detects the presence
of quiescent cells, and wherein the method further comprises analysing the nucleic acid
by performing sequence-based assays.
27
[0094] In an embodiment of the present disclosure, there is provided an in-vitro method
for detecting presence of quiescent cells, said method comprising: (a) obtaining a blood
sample; (b) enriching pluripotent stem cells from the blood sample, to obtain a mixture
comprising said pluripotent stem cells; (c) obtaining nucleic acid from the mixture of
step (b); (d) performing an assay with the nucleic acid for analysing 5 expression level
of at least one biomarker of pluripotent stem cell; and (e) comparing the expression
level of the at least one biomarker of pluripotent stem cell in the sample with an
expression level of the at least one biomarker in a control sample, wherein an increase
in the expression level of the at least one biomarker in the sample as compared to the
10 expression level of the at least one biomarker in the control sample detects the presence
of quiescent cells, and wherein the increase is at most 1.9 folds. In another embodiment,
the increase is in a range of 0.1-1.9 folds. In yet another embodiment, the increase is in
a range of 0.2-1.8 folds.
[0095] In an embodiment of the present disclosure, there is provided an in-vitro method
15 for detecting cancer, said method comprising: (a) obtaining a blood sample; (b)
enriching pluripotent stem cells from the blood sample, to obtain a mixture comprising
said pluripotent stem cells; (c) obtaining nucleic acid from the mixture of step (b); (d)
performing an assay with the nucleic acid for analysing expression level of at least one
biomarker of pluripotent stem cell; and (e) comparing the expression level of the at
20 least one biomarker of pluripotent stem cell in the sample with an expression level of
the at least one biomarker in a control sample, wherein an increase in the expression
level of the at least one biomarker in the sample as compared to the expression level of
the at least one biomarker in the control sample detects cancer.
[0096] In an embodiment of the present disclosure, there is provided an in-vitro method
25 for detecting cancer, said method comprising: (a) obtaining a blood sample; (b)
enriching pluripotent stem cells from the blood sample, to obtain a mixture comprising
said pluripotent stem cells; (c) obtaining nucleic acid from the mixture of step (b); (d)
performing an assay with the nucleic acid for analysing expression level of at least one
biomarker of pluripotent stem cell; and (e) comparing the expression level of the at
28
least one biomarker of pluripotent stem cell in the sample with an expression level of
the at least one biomarker in a control sample, wherein an increase in the expression
level of the at least one biomarker in the sample as compared to the expression level of
the at least one biomarker in the control sample detects cancer, and wherein the increase
in the expression level is in a range of 10-20 folds as 5 compared to the control and
detects stage-I cancer.
[0097] In an embodiment of the present disclosure, there is provided an in-vitro method
for detecting cancer, said method comprising: (a) obtaining a blood sample; (b)
enriching pluripotent stem cells from the blood sample, to obtain a mixture comprising
10 said pluripotent stem cells; (c) obtaining nucleic acid from the mixture of step (b); (d)
performing an assay with the nucleic acid for analysing expression level of at least one
biomarker of pluripotent stem cell; and (e) comparing the expression level of the at
least one biomarker of pluripotent stem cell in the sample with an expression level of
the at least one biomarker in a control sample, wherein an increase in the expression
15 level of the at least one biomarker in the sample as compared to the expression level of
the at least one biomarker in the control sample detects cancer, and wherein the increase
in the expression level is in a range of 20-30 folds as compared to the control and
detects stage-II cancer.
[0098] In an embodiment of the present disclosure, there is provided an in-vitro method
20 for detecting cancer, said method comprising: (a) obtaining a blood sample; (b)
enriching pluripotent stem cells from the blood sample, to obtain a mixture comprising
said pluripotent stem cells; (c) obtaining nucleic acid from the mixture of step (b); (d)
performing an assay with the nucleic acid for analysing expression level of at least one
biomarker of pluripotent stem cell; and (e) comparing the expression level of the at
25 least one biomarker of pluripotent stem cell in the sample with an expression level of
the at least one biomarker in a control sample, wherein an increase in the expression
level of the at least one biomarker in the sample as compared to the expression level of
the at least one biomarker in the control sample detects cancer, and wherein the increase
29
in the expression level is in a range of 30-40 folds as compared to the control and
detects stage-III cancer.
[0099] In an embodiment of the present disclosure, there is provided an in-vitro method
for detecting cancer, said method comprising: (a) obtaining a blood sample; (b)
enriching pluripotent stem cells from the blood sample, to obtain 5 a mixture comprising
said pluripotent stem cells; (c) obtaining nucleic acid from the mixture of step (b); (d)
performing an assay with the nucleic acid for analysing expression level of at least one
biomarker of pluripotent stem cell; and (e) comparing the expression level of the at
least one biomarker of pluripotent stem cell in the sample with an expression level of
10 the at least one biomarker in a control sample, wherein an increase in the expression
level of the at least one biomarker in the sample as compared to the expression level of
the at least one biomarker in the control sample detects cancer, and wherein the increase
in the expression level is 40 folds or higher as compared to the control and detects
stage-IV cancer.
15 [00100] In an embodiment of the present disclosure, there is provided an in-vitro
method for detecting cancer, said method comprising: (a) obtaining a blood sample; (b)
enriching pluripotent stem cells from the blood sample, to obtain a mixture comprising
said pluripotent stem cells; (c) obtaining nucleic acid from the mixture of step (b); (d)
performing an assay with the nucleic acid for analysing expression level of at least one
20 biomarker of pluripotent stem cell; and (e) comparing the expression level of the at
least one biomarker of pluripotent stem cell in the sample with an expression level of
the at least one biomarker in a control sample, wherein an increase in the expression
level of the at least one biomarker in the sample as compared to the expression level of
the at least one biomarker in the control sample detects cancer, and wherein the increase
25 in the expression level is in a range of 6-10 folds as compared to the control and detects
pre-cancerous stage.
[00101] In an embodiment of the present disclosure, there is provided an in-vitro
method for detecting cancer, said method comprising: (a) obtaining a blood sample; (b)
enriching pluripotent stem cells from the blood sample, to obtain a mixture comprising
30
said pluripotent stem cells; (c) obtaining nucleic acid from the mixture of step (b); (d)
performing an assay with the nucleic acid for analysing expression level of at least one
biomarker of pluripotent stem cell; and (e) comparing the expression level of the at
least one biomarker of pluripotent stem cell in the sample with an expression level of
the at least one biomarker in a control sample, wherein an increase 5 in the expression
level of the at least one biomarker in the sample as compared to the expression level of
the at least one biomarker in the control sample detects cancer, and wherein the method
further comprises analysing the nucleic acid by performing sequence-based assays. In
another embodiment of the present disclosure, analysing the nucleic acid by sequence10
based assays detects the type of cancer.
[00102] In an embodiment of the present disclosure, there is provided an in-vitro
method for detecting cancer, said method comprising: (a) obtaining a blood sample; (b)
enriching pluripotent stem cells from the blood sample, to obtain a mixture comprising
said pluripotent stem cells; (c) obtaining nucleic acid from the mixture of step (b); (d)
15 performing an assay with the nucleic acid for analysing expression level of at least one
biomarker of pluripotent stem cell; and (e) comparing the expression level of the at
least one biomarker of pluripotent stem cell in the sample with an expression level of
the at least one biomarker in a control sample, wherein an increase in the expression
level of the at least one biomarker in the sample as compared to the expression level of
20 the at least one biomarker in the control sample detects cancer, and wherein the increase
in the expression level of the at least one biomarker of pluripotent stem cell is at least
2 folds as compared to the control. In another embodiment, the increase in the
expression level of the at least one biomarker of pluripotent stem cell is at least 3 folds
as compared to the control. In yet another embodiment, the increase in the expression
25 level of the at least one biomarker of pluripotent stem cell is at least 5 folds as compared
to the control.
[00103] In an embodiment of the present disclosure, there is provided an in-vitro
method for detecting cancer, said method comprising: (a) obtaining a blood sample; (b)
enriching pluripotent stem cells from the blood sample, to obtain a mixture comprising
31
said pluripotent stem cells; (c) obtaining nucleic acid from the mixture of step (b); (d)
performing an assay with the nucleic acid for analysing expression level of at least one
biomarker of pluripotent stem cell; and (e) comparing the expression level of the at
least one biomarker of pluripotent stem cell in the sample with an expression level of
the at least one biomarker in a control sample, wherein an increase 5 in the expression
level of the at least one biomarker in the sample as compared to the expression level of
the at least one biomarker in the control sample detects cancer, and wherein the increase
in the expression level of the at least one biomarker of pluripotent stem cell is at least
6 folds, or 7 folds, or 8 folds, or 9 folds, or 10 folds as compared to the control.
10 [00104] In an embodiment of the present disclosure, there is provided an in-vitro
method for detecting cancer, said method comprising: (a) obtaining a blood sample; (b)
enriching pluripotent stem cells from the blood sample, to obtain a mixture comprising
said pluripotent stem cells; (c) obtaining nucleic acid from the mixture of step (b); (d)
performing an assay with the nucleic acid for analysing expression level of at least one
15 biomarker of pluripotent stem cell; and (e) comparing the expression level of the at
least one biomarker of pluripotent stem cell in the sample with an expression level of
the at least one biomarker in a control sample, wherein an increase in the expression
level of the at least one biomarker in the sample as compared to the expression level of
the at least one biomarker in the control sample detects cancer, and wherein the increase
20 in the expression level of the at least one biomarker of pluripotent stem cell is in a range
of 10-20 folds as compared to the control. In another embodiment, the increase in the
expression level of the at least one biomarker of pluripotent stem cell is in a range of
20-30 folds as compared to the control. In yet another embodiment, the increase in the
expression level of the at least one biomarker of pluripotent stem cell is in a range of
25 30-40 folds as compared to the control. In one another embodiment, the increase in the
expression level of the at least one biomarker of pluripotent stem cell is 40 folds or
higher as compared to the control.
[00105] In an embodiment of the present disclosure, there is provided an in-vitro
method for detecting cancer, said method comprising: (a) obtaining a blood sample; (b)
32
enriching pluripotent stem cells from the blood sample, to obtain a mixture comprising
said pluripotent stem cells; (c) obtaining nucleic acid from the mixture of step (b); (d)
performing an assay with the nucleic acid for analysing expression level of at least one
biomarker of pluripotent stem cell; and (e) comparing the expression level of the at
least one biomarker of pluripotent stem cell in the sample with 5 an expression level of
the at least one biomarker in a control sample, wherein an increase in the expression
level of the at least one biomarker in the sample as compared to the expression level of
the at least one biomarker in the control sample detects cancer, and wherein the at least
one biomarker of pluripotent stem cell is selected from a group consisting of Oct-4,
10 Sox-2, Nanog, p53, Sirt-1, Sirt-6, Sirt-3, NAD, RAS, ERC, erbB-2, ABL, subsets
thereof, and combinations thereof. In another embodiment, the at least one biomarker
of pluripotent stem cell is Oct-4, and subsets thereof. In yet another embodiment, the
at least one biomarker of pluripotent stem cell is Oct-4a. In one another embodiment,
the at least one biomarker of pluripotent stem cell is Oct-4b.
15 [00106] In an embodiment of the present disclosure, there is provided an in-vitro
method for detecting cancer, said method comprising: (a) obtaining a blood sample; (b)
enriching pluripotent stem cells from the blood sample, to obtain a mixture comprising
said pluripotent stem cells; (c) obtaining nucleic acid from the mixture of step (b); (d)
performing an assay with the nucleic acid for analysing expression level of at least one
20 biomarker of pluripotent stem cell; and (e) comparing the expression level of the at
least one biomarker of pluripotent stem cell in the sample with an expression level of
the at least one biomarker in a control sample, wherein an increase in the expression
level of the at least one biomarker in the sample as compared to the expression level of
the at least one biomarker in the control sample detects cancer, and wherein the at least
25 one biomarker of pluripotent stem cell is selected from a group consisting of Oct-4,
Sox-2, Nanog, p53, Sirt-1, Sirt-6, Sirt-3, subsets thereof, and combinations thereof. In
another embodiment, the at least one biomarker of pluripotent stem cell is Sox-2, and
subsets thereof. In yet another embodiment, the at least one biomarker of pluripotent
stem cell is Nanog, and subsets thereof. In one another embodiment, the at least one
33
biomarker of pluripotent stem cell is p53, and subsets thereof. In alternate embodiment,
the at least one biomarker of pluripotent stem cell is Sirt-1, and subsets thereof. In a
still alternate embodiment, the at least one biomarker of pluripotent stem cell is Sirt-6,
and subsets thereof. In one another alternate embodiment, the at least one biomarker of
pluripotent stem cell 5 is Sirt-3, and subsets thereof.
[00107] In an embodiment of the present disclosure, there is provided an in-vitro
I/We Claim:
1. An in-vitro method for detecting presence of metabolically altered cells, said
method comprising:
a) obtaining a blood sample;
b) enriching pluripotent stem cells from 5 the blood sample, to obtain a
mixture comprising said pluripotent stem cells;
c) obtaining nucleic acid from the mixture of step (b);
d) performing an assay with the nucleic acid for analysing expression level
of at least one biomarker of pluripotent stem cell; and
10 e) comparing the expression level of the at least one biomarker of
pluripotent stem cell in the sample with an expression level of the at
least one biomarker in a control sample,
wherein an increase in the expression level of the at least one biomarker in the
sample as compared to the expression level of the at least one biomarker in the
15 control sample detects the presence of metabolically altered cells.
2. An in-vitro method for detecting presence of quiescent cells, said method
comprising:
a) obtaining a blood sample;
b) enriching pluripotent stem cells from the blood sample, to obtain a
20 mixture comprising said pluripotent stem cells;
c) obtaining nucleic acid from the mixture of step (b);
d) performing an assay with the nucleic acid for analysing expression level
of at least one biomarker of pluripotent stem cell; and
e) comparing the expression level of the at least one biomarker of
25 pluripotent stem cell in the sample with an expression level of the at
least one biomarker in a control sample,
86
wherein an increase in the expression level of the at least one biomarker
in the sample as compared to the expression level of the at least one
biomarker in the control sample detects the presence of quiescent cells.
3. An in-vitro method for detecting cancer, said method comprising:
5 a) obtaining a blood sample;
b) enriching pluripotent stem cells from the blood sample, to obtain a
mixture comprising said pluripotent stem cells;
c) obtaining nucleic acid from the mixture of step (b);
d) performing an assay with the nucleic acid for analysing expression level
10 of at least one biomarker of pluripotent stem cell; and
e) comparing the expression level of the at least one biomarker of
pluripotent stem cell in the sample with an expression level of the at
least one biomarker in a control sample,
wherein an increase in the expression level of the at least one biomarker
15 in the sample as compared to the expression level of the at least one
biomarker in the control sample detects cancer.
4. An in-vitro method for predicting cancer, said method comprising:
a) obtaining a blood sample;
b) enriching pluripotent stem cells from the blood sample, to obtain a
20 mixture comprising said pluripotent stem cells;
c) obtaining nucleic acid from the mixture of step (b);
d) performing an assay with the nucleic acid for analysing expression level
of at least one biomarker of pluripotent stem cell; and
e) comparing the expression level of the at least one biomarker of
25 pluripotent stem cell in the sample with an expression level of the at
least one biomarker in a control sample,
87
wherein an increase in the expression level of the at least one biomarker in
the sample as compared to the expression level of the at least one biomarker
in the control sample predicts cancer.
5. The method as claimed in any one of the claims 1-4, wherein the method further
comprises analysing the nucleic acid by performing 5 sequence-based assays.
6. The method as claimed in claim 5, wherein analysing the nucleic acid by
sequence-based assays detects the type of cancer.
7. The method as claimed in any one of the claims 1, 3, or 4, wherein the increase
in the expression level of the at least one biomarker of pluripotent stem cell is
10 at least 2 folds as compared to the control.
8. The method as claimed in any one of the claims 1, 3, or 4, wherein the increase
in the expression level of the at least one biomarker of pluripotent stem cell is
at least 3 folds as compared to the control.
9. The method as claimed in any one of the claims 1, 3, or 4, wherein the increase
15 in the expression level of the at least one biomarker of pluripotent stem cell is
at least 5 folds as compared to the control.
10. The method as claimed in any one of the claims 1, 3, or 4, wherein the increase
in the expression level of the at least one biomarker of pluripotent stem cell is
in a range of 10-20 folds as compared to the control.
20 11. The method as claimed in any one of the claims 1, 3, or 4, wherein the increase
in the expression level of the at least one biomarker of pluripotent stem cell is
in a range of 20-30 folds as compared to the control.
12. The method as claimed in any one of the claims 1, 3, or 4, wherein the increase
in the expression level of the at least one biomarker of pluripotent stem cell is
25 in a range of 30-40 folds as compared to the control.
13. The method as claimed in any one of the claims 1, 3, or 4, wherein the increase
in the expression level of the at least one biomarker of pluripotent stem cell is
in a range of 40-50 folds as compared to the control.
88
14. An in-vitro method for monitoring response to anti-cancer therapy, said method
comprising:
a) obtaining a blood sample at one time point during an anti-cancer
therapy;
b) enriching pluripotent stem cells from 5 the blood sample to obtain a
mixture comprising said pluripotent stem cells;
c) obtaining nucleic acid from the mixture of step (b);
d) performing an assay with the nucleic acid for analysing expression level
of at least one biomarker of pluripotent stem cell; and
10 e) comparing the expression level of the at least one biomarker of
pluripotent stem cell in the sample with an expression level of the at
least one biomarker of pluripotent stem cell in a reference that monitors
the response to anti-cancer therapy.
15. The method as claimed in claim 14, wherein the reference is at least one
15 selected from a group consisting of: (a) a blood sample obtained prior to
administration of anti-cancer therapy; (b) a blood sample obtained at a previous
time point as compared to the time point mentioned in step (a) of claim 14; (c)
a blood sample obtained at a subsequent time point as compared to the time
point mentioned in step (a) of claim 14; and (d) a blood sample obtained from
20 a cancer-free subject.
16. The method as claimed in claim 14, wherein a decrease in the expression level
of the at least one biomarker of pluripotent stem cell as compared to the
expression level in the reference indicates a positive response to the anti-cancer
therapy, and wherein the reference is at least one selected from a group
25 consisting of: (a) a blood sample obtained prior to administration of anti-cancer
therapy; (b) a blood sample obtained at a previous time point as compared to
the time point mentioned in step (a) of claim 14; and (c) a blood sample
obtained from a cancer-free subject.
89
17. An in-vitro method for detecting a positive response to anti-cancer therapy,
said method comprising:
a) obtaining a blood sample-I before administration of an anti-cancer
therapy;
b) obtaining a blood sample-II after administration 5 of the anti-cancer
therapy;
c) enriching pluripotent stem cells from the blood sample-I to obtain a
mixture-I comprising said pluripotent stem cells;
d) enriching pluripotent stem cells from the blood sample-II to obtain a
10 mixture-II comprising said pluripotent stem cells;
e) obtaining nucleic acid-I from the mixture-I;
f) obtaining nucleic acid-II from the mixture-II;
g) independently performing an assay with the nucleic acid-I and the
nucleic acid-II for analysing expression level of at least one biomarker
15 of pluripotent stem cell; and
h) comparing the expression levels of the at least one biomarker of
pluripotent stem cell from the nucleic acid-II with the expression level
of the at least one biomarker of pluripotent stem cell from the nucleic
acid-I,
20 wherein a decrease in the expression level of the at least one biomarker
of pluripotent stem cell from the nucleic acid-II as compared to the
expression level of the at least one biomarker of pluripotent stem cell from
the nucleic acid-I detects a positive response to the cancer treatment.
18. An in-vitro method for detecting cancer, said method comprising:
25 a) obtaining a blood sample;
b) enriching pluripotent stem cells from the blood sample, to obtain a
mixture comprising said pluripotent stem cells;
c) obtaining nucleic acid from the mixture of step (b);
90
d) performing an assay with the nucleic acid for analysing expression level
of at least one biomarker of pluripotent stem cell;
e) comparing the expression level of the at least one biomarker of
pluripotent stem cell in the sample with an expression level of the at
least one biomarker in a control sample, wherein 5 an increase in the
expression level of the at least one biomarker in the sample as compared
to the expression level of the at least one biomarker in the control
sample indicates presence of cancer; and
f) performing sequence-based assays on the nucleic acid and analysing for
10 mutation in at least one cancer-related marker,
wherein presence of mutation in the at least one cancer-related marker
indicates presence of a specific type of cancer based on the cancerrelated
marker analysed.
19. The method as claimed in any one of the claims 1-4, 14, 17, or 18, wherein the
15 at least one biomarker of pluripotent stem cell is selected from a group
consisting of Oct-4, Sox-2, Nanog, p53, Sirt-1, Sirt-6, Sirt-3, NAD, RAS, ERC,
erbB-2, ABL, subsets thereof, and combinations thereof.
20. The method as claimed in any one of the claims 1-4, 14, 17, or 18, wherein
obtaining the nucleic acid from the mixture is by any one method selected from
20 a group consisting of: (a) guanidinium thiocyanate-phenol-chloroform nucleic
acid extraction; (b) cesium chloride gradient centrifugation method; (c)
cetyltrimethylammonium bromide nucleic acid extraction; (d) alkaline
extraction; (e) resin-based extraction; and (f) solid phase nucleic acid
extraction.
25 21. The method as claimed in any one of the claims 1-4, 14, 17, or 18, wherein
performing an assay with the nucleic acid for analysing the expression of the at
least one biomarker is done by a technique selected from a group consisting of:
quantitative PCR, flow cytometry, and Next Generation Sequencing (NGS).
91
22. The method as claimed in any one of the claims 1-4, 14, or 18, wherein the
control is the expression level of the at least one biomarker of pluripotent stem
cells obtained from a cancer-free subject.
23. The method as claimed in any one of the claims 1-4, 14, 17, or 18, wherein the
enriching of the pluripotent stem cells f 5 rom the blood sample comprises:
a) contacting the blood sample with a neutral buffer in a ratio range of 1:1
to 1:20, to obtain a first mixture;
b) contacting at least one salt solution to the first mixture in a ratio range
of 1:2 to 1:10, to obtain a second mixture; and
10 c) processing the second mixture to obtain enriched pluripotent stem cells.
24. The method as claimed in claim 23, wherein the processing of the second
mixture comprises, at least one method selected from a group consisting of: (a)
extraction process; (b) washing process; (c) centrifugation process, and
combinations thereof.
15 25. The method as claimed in claim 18, wherein the cancer-related marker is
selected from a group consisting of well-known markers established to be
related to cancer.
26. The method as claimed in any one of the claims 1-4, 14, 17, or 18, wherein the
method is independent of invasive techniques.
20 27. Use of a pluripotent stem cell biomarker selected from a group consisting of
Oct-4, Sox-2, Nanog, p53, NFκB, Sirt-1, Sirt-6, Sirt-3, NAD, RAS, ERC, erbB-
2, ABL, subsets thereof, and combinations thereof, for detecting cancer from a
blood sample.
28. Use of a pluripotent stem cell biomarker selected from a group consisting of
25 Oct-4, Sox-2, Nanog, p53, NFκB, Sirt-1, Sirt-6, Sirt-3, NAD, RAS, ERC, erbB-
2, ABL, subsets thereof, and combinations thereof, for predicting cancer from
a blood sample.
29. Use of a pluripotent stem cell biomarker selected from a group consisting of
Oct-4, Sox-2, Nanog, p53, NFκB, Sirt-1, Sirt-6, Sirt-3, NAD, RAS, ERC, erbB-
92
2, ABL, subsets thereof, and combinations thereof, for grading stage of cancer
from a blood sample.
30. Use of a pluripotent stem cell biomarker selected from a group consisting of
Oct-4, Sox-2, Nanog, p53, NFκB, Sirt-1, Sirt-6, Sirt-3, NAD, RAS, ERC, erbB-
2, ABL, subsets thereof, and combinations thereof, 5 for monitoring progression
of anti-cancer therapy from a blood sample.
31. A method for treating cancer, said method comprising:
a) obtaining a blood sample from a subject;
b) enriching pluripotent stem cells from the blood sample, to obtain a
10 mixture comprising said pluripotent stem cells;
c) obtaining nucleic acid from the mixture of step (b);
d) performing an assay with the nucleic acid for analysing expression level
of at least one biomarker of pluripotent stem cell;
e) comparing the expression level of the at least one biomarker of
15 pluripotent stem cell in the sample with an expression level of the at
least one biomarker in a control sample, wherein an increase in the
expression level of the at least one biomarker in the sample as compared
to the expression level of the at least one biomarker in the control
sample detects cancer; and
20 f) administering anti-cancer therapy to the subject for treating can