US-12618114-B2 - Method for identifying cancer in a subject

Abstract

The present disclosure discloses an in-vitro and non-invasive method for detecting a medical condition in a subject. The method involves enriching very small embryonic like stem cells from the sample, to obtain a mixture comprising said very small embryonic like stem cells; obtaining nucleic acid from the mixture of step; performing an assay with the nucleic acid for analysing expression level of Oct4A in the very small embryonic like stem cells from the sample; and comparing the expression level of Oct4A in the very small embryonic like stem cells from the sample with an expression level of Oct4A in a control sample. The present disclosure also provides a method for predicting the onset of cancer and for predicting the presence of cancer. A method of treating cancer is also disclosed herein. Moreover, a reagent kit and a detection kit are also disclosed.

Inventors

• Ashish Tripathi

Assignees

• 23IKIGAI PTE LTD

Dates

Publication Date

20260505

Application Date

20210507

Claims (16)

1. 1 . A method for detecting a presence or an absence of cancer in a subject, comprising: (a) obtaining a whole blood sample from the subject; (b) contacting the whole blood sample with a neutral buffer and then with a salt solution, followed by performing a first centrifugation at a first speed to obtain a first pellet of cells, wherein the contacting is performed with a first ratio range of 1:1 to 1:20 between the whole blood sample and the neutral buffer, and wherein the contacting is performed with a second ratio in a range of 1:2 to 1:10 between (1) the salt solution and (2) the whole blood sample contacted with the neutral buffer; (c) performing a second centrifugation at a second speed to obtain a second pellet of cells; (d) isolating messenger ribonucleic acid (mRNA) from the second pellet of cells; (e) assaying the isolated mRNA to (i) detect a presence of Oct4A-expressing cells among the second pellet of cells and (ii) determine an expression level of Oct4A; (f) comparing the determined expression level of Oct4A with a reference expression level; and (g) detecting the presence of cancer in the subject when the determined expression level of Oct4A is increased by a range of at least 5 folds and less than 10 folds as compared to the reference expression level.
2. 2 . A method for predicting onset of cancer in a subject, comprising: (a) obtaining a whole blood sample from the subject; (b) contacting the whole blood sample with a neutral buffer and then with a salt solution, followed by performing a first centrifugation to obtain a first pellet of cells, wherein the contacting is performed with a first ratio range of 1:1 to 1:20 between the whole blood sample and the neutral buffer, and wherein the contacting is performed with a second ratio in a range of 1:2 to 1:10 between (1) the salt solution and (2) the whole blood sample contacted with the neutral buffer; (c) performing a second centrifugation at a second speed to obtain a second pellet of cells; (d) isolating messenger ribonucleic acid (mRNA) from the second pellet of cells; (e) assaying the isolated mRNA to (i) detect a presence of Oct4A-expressing cells among the second pellet of cells and (ii) determine an expression level of Oct4A; (f) comparing the determined expression level of Oct4A with a reference expression level; and (g) predicting the onset of cancer in the subject when the determined expression level of Oct4A is increased by a range of at least 3 folds and less than 5 folds as compared to the reference expression level.
3. 3 . The method of claim 1 , wherein the assaying comprises sequencing the isolated mRNA or derivatives thereof to determine the expression level of Oct4A.
4. 4 . The method of claim 2 , wherein the assaying comprises sequencing the isolated mRNA or derivatives thereof to determine the expression level of Oct4A.
5. 5 . The method of claim 1 , wherein (d) further comprises at least one of: (a) guanidinium thiocyanate-phenol-chloroform nucleic acid extraction; (b) cesium chloride gradient centrifugation; (c) cetyltrimethylammonium bromide nucleic acid extraction; (d) alkaline extraction; (e) resin-based extraction; and (f) solid phase nucleic acid extraction.
6. 6 . The method of claim 2 , wherein (d) further comprises at least one of: (i) guanidinium thiocyanate-phenol-chloroform nucleic acid extraction; (ii) cesium chloride gradient centrifugation; (iii) cetyltrimethylammonium bromide nucleic acid extraction; (iv) alkaline extraction; (v) resin-based extraction; and (vi) solid phase nucleic acid extraction.
7. 7 . The method of claim 1 , wherein the reference expression level of Oct4A is determined from a whole blood sample of a cancer-free subject.
8. 8 . The method of claim 1 , wherein the method is independent of invasive techniques.
9. 9 . The method of claim 2 , wherein (e) further comprises performing nucleic acid sequencing of the isolated mRNA to detect a presence or an absence of a mutation in at least one cancer-related genetic marker.
10. 10 . The method of claim 2 , wherein the method is independent of invasive techniques.
11. 11 . The method of claim 1 , wherein the assaying comprises performing quantitative polymerase chain reaction (qPCR).
12. 12 . The method of claim 2 , wherein the assaying comprises performing qPCR.
13. 13 . The method of claim 3 , further comprising reverse transcribing the isolated mRNA to produce complementary deoxyribonucleic acid (cDNA), and sequencing the cDNA or derivatives thereof to determine the expression level of Oct4A.
14. 14 . The method of claim 4 , further comprising reverse transcribing the isolated mRNA to produce complementary deoxyribonucleic acid (cDNA), and sequencing the cDNA or derivatives thereof to determine the expression level of Oct4A.
15. 15 . The method of claim 1 , wherein the first centrifugation comprises density gradient centrifugation using Ficoll-Hypaque.
16. 16 . The method of claim 2 , wherein the first centrifugation comprises density gradient centrifugation using Ficoll-Hypaque.

Description

CROSS-REFERENCE This application claims the benefit of U.S. Provisional Application No. 63/041,413, filed Jun. 19, 2020, U.S. Provisional Application No. 63/109,073, filed Nov. 3, 2020, each of which is incorporated herein by reference in their entireties. INCORPORATION BY REFERENCE OF MATERIAL SUBMITTED ELECTRONICALLY The Sequence Listing, which is a part of the present disclosure, is submitted concurrently with the specification as a text file. The name of the text file containing the Sequence Listing is “56773_Seqlisting.txt”, which was created on May 6, 2021 and is 2,336 bytes in size. The subject matter of the Sequence Listing is incorporated herein in its entirety by reference. FIELD OF THE INVENTION The present disclosure broadly relates to the field healthcare technologies, and particularly provides a simplified method for detecting the presence or absence of a medical condition in a human subject. The method as disclosed herein also detects for the presence or absence of an inflammatory condition in the human subject from the blood sample. Further, the method as described in the present disclosure, also detects the presence, or absence, or imminent presence of cancer in a subject. The method as described herein is an in-vitro method which involves analysing the sample obtained from a human subject. BACKGROUND OF INVENTION Research on the genetic causes of disease has accelerated as a result of both the completion of the human genome and the development of the Next Generation Sequencing techniques, which has opened the promise of translating the alterations in individuals' genomes in clinically relevant information to assist disease diagnostics and therapeutic, clinical decision-making strategies. These efforts have generated a large volume of potentially useful information in form of enormous amounts of data that has boosted biomedical research. Application and interpretation of this information, however, is still cumbersome and time-consuming for researchers, because the clinically relevant molecular fingerprint of the mutation profiles is derived out of tissues extracted from biopsy procedures. Biopsy is a well-known technique which involves removal of tissue under examination for disease diagnosis and further treatment approaches. Usually, a biopsy is invasive, and involves complex, surgical procedures for removal of tissue from their native environment. Tissue biopsy is the “gold standard” for cancer, but interestingly, a number of non-cancerous tissues (i.e. diseased tissues) are also excised in order to detect the origin, transmission, progression of disease etc. that dilutes the original disease data and leads to false positives including misdiagnosis. Almost all tissues can be studied through biopsy including muscle, thyroid, bladder, heart, prostate, skin, lung, lymph node, liver, kidney, nerves etc. Some diseases for which biopsies are included in the scientific literature are cortical demyelination in brain white matter lesions for early detection of multiple sclerosis (Lucchinetti et. al. 2011), percutaneous renal biopsy for kidney diseases, cirrhotic liver disease, hepatitis C-associated glomerulonephritis and cryoglobulinemic vasculitis, monoclonal gammopathy etc. (Hogan, Mocanu, and Berns 2016), synovial biopsy for detection of mononuclear infiltrates, fibrosis, angiogenesis, macrophage infiltration and lining layer thickening in tissues of osteoarthritis patients (Ene et al. 2015), shave, punch or incisional biopsy for inflammatory skin disorders (Harvey, Chan, and Wood 2017), computer-tomography guided lung biopsy for evaluation of COPD (Asai et al. 2013), myocardial biopsy (Francis and Lewis 2018), liver biopsy for cirrhotic patients (Sherman et al. 2007) etc. However, most tissue biopsies result in surgical complications, bleeding, and adverse side-effects etc. and hence are not recommended as opposed to biofluid tests such as of blood, urine, saliva etc. Tissue biopsies are difficult to perform, resulting in painful, often discomfort procedures that may not identify the exact anatomical location of the tumor or may further cause metastasis-promoting complications due to surgical excision of angiogenesis-rich areas. Owing to the complexities of the tissue biopsy procedure and mixed results obtained, and the lack of clarity associated with such studies with respect to the tissue to be studied vis-à-vis the condition of a subject, there is a knowledge gap which exists in this area of work. Stem cells, particularly of embryonic origin, possess pluripotency markers viz. Oct4, Nanog, Sox2 and their isoforms are indicative of varied differentiation potentials into multiple tissues forming organs in development, homeostasis and aging. Since stem cells contribute to tissue development, they act as molecular biosensors implicative of tissue damage and injury, a hallmark of medical conditions. Thus, stem cell markers are prominent biomarkers for determining severity of medical conditions a