Method for analysing pluripotent stem cell biomarkers, and implementations thereof

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 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 is at least 10 folds as compared to the expression level of the at least one biomarker in the control sample for detecting cancer.

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 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 the at least one biomarker in the control sample is at least 6 folds for predicting cancer.

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 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 expression level of the at least one biomarker of pluripotent stem cell in a reference that monitors the response to anti-cancer therapy, 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 is at least 2 folds for indicating a positive response to the anti-cancer therapy, and wherein the reference is at least one selected from the group consisting of:
• (i) a blood sample obtained prior to administration of anti-cancer therapy;
• (ii) a blood sample obtained at a previous time point as compared to the time point mentioned in step (a);
• and (iii) a blood sample obtained from a cancer-free subject.

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 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 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 is at least 6 folds for detecting cancer;
• and
• f) administering anti-cancer therapy to the subject for treating cancer,
• wherein the enriching of the pluripotent stem cells from the blood sample comprises:
• (i) contacting the blood sample with a neutral buffer in a ratio range of 1:1 to 1:20, to obtain a first mixture;
• (ii) 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 (iii) processing the second mixture to obtain enriched pluripotent stem cells, wherein the processing of the second mixture comprises:
• (i) extraction process;
• (ii) washing process;
• (iii) centrifugation process, and combinations thereof, wherein the at least one biomarker of pluripotent stem cell is selected from the group consisting of Oct-4, Oct-4a, Oct-4b, Oct 4b1, Sox-2, Nanog, p53, Sirt-1, Sirt-6, Sirt-3, NAD, RAS, ERC, erbB-2, ABL, subsets thereof, and combinations thereof, and wherein obtaining the nucleic acid from the mixture is by any one method selected from the 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, and 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 the group consisting of:
• quantitative PCR, flow cytometry, and Next Generation Sequencing (NGS).