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1. WO2018102162 - METHODS AND COMPOSITIONS TO DETECT MUTATIONS IN PLASMA USING EXOSOMAL RNA AND CELL FREE DNA FROM NON-SMALL CELL LUNG CANCER PATIENTS

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[ EN ]

What is claimed is:

1. A method for the diagnosis, prognosis, monitoring or therapy selection for a disease or other medical condition in a subject in need thereof, the method comprising the steps of:

(a) providing a biological sample from a subject;

(b) isolating microvesicles from the biological sample;

(c) extracting one or more nucleic acids from the microvesicles;

(d) detecting a T790M mutation, an L858R mutation, one or more exon19 insertions and/or one or more exon19 deletions in the Epidermal Growth Factor Receptor (EGFR) gene in the extracted nucleic acids to obtain a value(s), and

(e) comparing the value(s) to a pre-defined cutoff threshold(s) to determine the presence or the absence of the T790M mutation, the L858R mutation, the one or more exon19 insertions and/or the one or more exon19 deletions in the extracted nucleic acids,

wherein the presence of the T790M mutation, the L858R mutation, the one or more exon19 insertions and/or the one or more exon19 deletions in the extracted nucleic acids indicates the presence of a disease or other medical condition in the subject or a higher predisposition of the subject to develop a disease or other medical condition.

2. The method of claim 1, wherein the value obtained in step (d) is a cycle threshold (Ct) value.

3. The method of claim 1, wherein step (c) further comprises the co-isolation of extracellular nucleic acids and circulating nucleic acids from the biological sample.

4. The method of claim 3, wherein step (c) further comprises reverse transcription of the one or more nucleic acids extracted from the microvesicles, the co-isolated extracellular nucleic acids and the circulating nucleic acids.

5. The method of claim 4, wherein the reverse transcription step comprises the use of one or more amplification controls.

6. The method of claim 5, wherein the amplification control is a nucleic acid, such as DNA.

7. The method of claim 4, wherein the reverse transcription step comprises the use of one or more controls of inhibition.

8. The method of claim 7, wherein the control of inhibition is an exogenous nucleic acid.

9. The method of claim 8, wherein the exogenous nucleic acid is spiked into the reverse transcription step or at the extraction step at a known quantity.

10. The method of any one of claims 2 to 9, wherein the method further comprises after step (c) and prior to step (d) a pre-amplification reaction step.

11. The method of claim 10, wherein the pre-amplification reaction step is a multiplex pre-amplification reaction step.

12. The method of claim 10, wherein the pre-amplification reaction step is a single-plex pre-amplification reaction step.

13. The method of any one of claims 10 to 12, wherein the pre-amplification reaction step is performed under condition PCR conditions that favor pre-amplification of a mutant EGFR 20 nucleic acid sequence over wild type EGFR nucleic acid sequence.

14. The method of any one of claims 10 to 13, wherein the multiplex pre-amplification reaction comprises a wild type blocker for exon 19, exon 20, and/or exon 21 of EGFR.

15. The method of claim 14, wherein the wild type blocker is a hydrophobic nucleic acid, a bridge nucleic acid, a peptide nucleic acid, any oligonucleotide with a 3' end terminator, or any other modified base/nucleotide/sequence or condition that prevents an efficient amplification of the wild type sequence or combinations thereof.

16. The method of any one of claims 2 to 15, wherein step (d) comprises a sequencing-based detection technique, such as a PCR technique or a next-generation sequence technique, or any other PCR-based or PCR-free detection method.

17. The method of claim 16, wherein step (d) comprises qPCR.

18. The method of claim 17, wherein the qPCR is based on a mutant-specific amplification system or other mutation-biased amplification system.

19. The method of claim 18, wherein the qPCR is based on an Amplification Refractory Mutation system (ARMS).

20. The method of claim 20, wherein the ARMS qPCR step comprises a primer comprising a modified nucleotide, base or sequence, a probe comprising a modified nucleotide, base or sequence, or both a primer comprising a modified nucleotide, base or sequence and a probe comprising a modified nucleotide.

21. The method of claim 20, wherein the primer comprises a base modification selected from the group consisting of 2-aminopurine, 8-amino-2'-deoxyadenosine, trimetroxystilbene, C-5 propynyl-deoxycytidine, C-5 propynyl-deoxyuridine, 2-amino-2'-deoxyadenosine-5'-triphosphate, 2,6-diaminopurine (2-amino-dA), inverted dT, inverted dideoxy-T, hydroxymethyl dC, iso-dC, 5-methyl dC, aminoethyl-phenoxazine-deoxycytidine, and locked nucleic acids (LNA's), and the inclusion of at least one mismatched base at one of the bases to increase the nucleic acid interaction at the 3' end of the mutant specific prime, and a combination thereof.

22. The method of any one of claims 5 to 21, wherein step (d) further comprises detecting one or more of a control molecule.

23. The method of any one of claims 5 to 22, wherein step (d) further comprises detecting one or more mutations through a pre-determined cutoff threshold(s).

24. The method of any one of claims 2 to 22, wherein the method in step (e) further comprising analyzing the data using machine-learning based modeling, data mining methods, and/or statistical analysis to derive a pre-determined cutoff threshold(s).

25. The method of claim 24, wherein the data is analyzed to identify or predict disease outcome of the patient.

26. The method of claim 24, wherein the data is analyzed to stratify the patient within a patient population.

27. The method of claim 24, wherein the data is analyzed to identify or predict whether the patient is sensitive or resistant to treatment with an anti-EGFR therapy.

28. The method of claim 27, wherein the anti-EGFR therapy comprises treatment with an EGFR inhibitor.

29. The method of claim 24, wherein the data is analyzed to measure progression-free survival progress of the subject.

30. The method of claim 14, wherein the data is analyzed to select a treatment option for the subject when the T790M mutation, the L858R mutation, the one or more exon19 insertions and/or the one or more exon19 deletions is detected.

31. The method of claim 30, wherein the treatment option is treatment with a second or third generation tyrosine kinase EGFR inhibitor or any other drug that targets T790M, L858R, the one or more exon19 insertions and/or the one or more exon19 deletions.

32. The method of claim 29, wherein the method further comprises administering to the subject a therapeutically effective amount of a second or third generation tyrosine kinase EGFR inhibitor or any other drug that specifically targets T790M, L858R, the one or more exon 19 insertions and/or the one or more exon 19 deletions or combinations thereof.

33. The method of any one of claims 1 to 30, wherein the biological sample is a bodily fluid.

34. The method of claim 33, wherein the biological sample is plasma or serum.

35. The method of any one of claims 1 to 34, wherein the disease or other medical condition is cancer.

36. The method of claim 35, wherein the disease or other medical condition is lung cancer.

37. The method of claim 35, wherein the disease or other medical condition is non-small cell lung cancer (NSCLC).

38. The method of claim 1, wherein the extracted nucleic acid is an extracellular nucleic acid.

39. The method of claim 1, wherein the extracted nucleic acid is circulating nucleic acids (for example: necrotic DNA, or a combination of cell free DNA and necrotic DNA).

40. The method of claim 1, wherein the extracted nucleic acid is a combination of extracellular nucleic acids and circulating nucleic acids, a combination of RNA and necrotic DNA, a combination of cell free DNA and necrotic DNA, or a combination of RNA, cell free DNA and necrotic DNA.

41. The method of claim 1, wherein the step (d) is performed using an oligonucleotide sequence at least 90% identical to a sequence identified in Table 1.

42. The method of claim 1, wherein the step (d) is performed using an oligonucleotide blocker at least 90% identical to a sequence identified in Table 1.