# US 10,865,451 B2 · Non-invasive blood based monitoring of genomic alterations in cancer > Agents: this data is available as JSON from the patentgrep API and MCP server — see https://patentgrep.com/llms.txt - Number: US 10,865,451 B2 - Document ID: US10865451B2 - Status: Granted - Kind: B2 - Application number: 14/897,269 - Filed: 2014-06-11 - Published: 2016-05-19 - Granted: 2020-12-15 - Earliest claimed: 2013-06-11 - Assignees: Dana-Farber Cancer Institute, Inc. - Applicants: Dana-Farber Cancer Institute, Inc. - Inventors: Pasi A. Janne; Cloud P. Paweletz; Geoffrey Oxnard; Yanan Kuang - Web page: https://patentgrep.com/patent/US10865451B2 ## CPC - C12Q 1/6886: for cancer (immunoassay for cancer G01N33/575) - A61K 31/437: the heterocyclic ring system containing a five-membered ring having nitrogen as a ring hetero atom, e.g. indolizine, beta-carboline - A61K 31/517: ortho- or peri-condensed with carbocyclic ring systems, e.g. quinazoline, perimidine - C12Q 1/6827: for detection of mutation or polymorphism - C12Q 2600/106: Pharmacogenomics, i.e. genetic variability in individual responses to drugs and drug metabolism - C12Q 2600/156: Polymorphic or mutational markers - C12Q: MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS (immunoassay G01N33/53); COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES - A61K: PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES (devices or methods specially adapted for bringing pharmaceutical products into particular physical or administering forms A61J3/00; chemical aspects of, or use of materials for deodorisation of air, for disinfection or sterilisation, or for bandages, dressings, absorbent pads or surgical articles A61L; soap compositions C11D) ## Abstract The invention provides methods to monitor cell free nucleic acids. The method comprises obtaining a plasma sample from a subject known to have a cancer characterized by a pair of mutually exclusive mutations specific to the cancer; isolating cell free nucleic acids from the plasma sample obtained from the subject; measuring the amount a housekeeping gene and/or total DNA in the cell free nucleic acids isolated from the plasma sample to confirm that the amount of housekeeping gene and/or total DNA in the sample is within a selected range; measuring the amount of a first of the pair of mutually exclusive mutations specific to the cancer in the cell free nucleic acids isolated from the plasma sample; and indicating in a report that the subject has the first mutation when (a) the amount of the housekeeping gene and/or total DNA in the cell free nucleic acids isolated from the plasma sample is within the selected range and (b) the amount of the first mutation is increased as compared to a control amount, wherein the control amount is determined by measuring the apparent amount of the first mutation in control cell free nucleic acids isolated from plasma samples obtained from control subjects known to have the second of the pair of mutually exclusive mutations specific to the cancer using measuring conditions substantially the same as those used to measure the amount of the first mutation in the cell free nucleic acids isolated from the plasma sample from the subject. ## Claims ### Claim 1 A method to monitor cell free DNA comprising: (i) obtaining a body fluid sample from a subject known to have a cancer characterized by a pair of mutually exclusive mutations specific to the cancer; (ii) isolating cell free nucleic acids from the body fluid sample obtained from the subject; (iii) measuring the level of Line 1 in the cell free nucleic acids isolated from the body fluid sample, wherein the level of Line 1 in the sample is within a range of 3,000-700,000 pg/μL; (iv) measuring the amount of the first of the pair of mutually exclusive mutations specific to the cancer in the cell free nucleic acids isolated from the body fluid sample; and (v) indicating in a report that the subject has the first of the pair of mutually exclusive mutations when (a) the level of Line 1 in the cell free nucleic acids isolated from the body fluid sample is within the range of 3,000-700,000 pg/μL and (b) the amount of the first of the pair of mutually exclusive mutations is increased as compared to a control amount, wherein the control amount is the apparent amount of the first of the pair of mutually exclusive mutations in control cell free nucleic acids isolated from body fluid samples obtained from control subjects known to have the second of the pair of mutually exclusive mutations specific to the cancer using measuring conditions substantially the same as those used to measure the amount of the first of the pair of mutually exclusive mutations in the cell free nucleic acids isolated from the body fluid sample from the subject. ### Claim 2 (depends on 1) The method of claim 1, wherein the measuring of: the first of the pair of mutually exclusive mutations specific to the cancer in the cell free nucleic acids isolated from the body fluid sample obtained from the subject is performed by quantitative PCR, microarrays, Next-generation sequencing, chemiluminescence methods, fluorescent methods, digital detection, or mass spectrometry (MALDI-TOF). ### Claim 3 (depends on 1) The method of claim 1, wherein the cancer is lung cancer or colon cancer. ### Claim 4 (depends on 3) The method of claim 3, wherein the pair of mutually exclusive mutations is an epidermal growth factor receptor (EGFR) mutation and a Rat sarcoma (RAS) mutation, or wherein the pair of mutually exclusive mutations comprises an epidermal growth factor receptor (EGFR) mutation and a v-Ki-ras2 Kirsten rat sarcoma viral oncogene homolog (KRAS) mutation, or wherein the pair of mutually exclusive mutations comprises a v-raf murine sarcoma viral oncogene homolog B1 (BRAF) mutation and a Rat sarcoma (RAS) mutation. ### Claim 5 (depends on 4) The method of claim 4, wherein the EGFR mutation is selected from the group consisting of: L858R, T790M, L861Q, G719S, del 19 and exon 20 insertions. ### Claim 6 (depends on 4) The method of claim 4, wherein the KRAS mutation is G12C. ### Claim 7 (depends on 1) The method of claim 1, wherein the amount of the first of the pair of mutually exclusive mutations specific to the cancer is measured by digital droplet PCR. ### Claim 8 (depends on 1) The method of claim 1, wherein the amount of the first of the pair of mutually exclusive mutations is measured before and after administration of an anti-cancer therapy to the subject. ### Claim 9 (depends on 1) The method of claim 1, wherein step (i) step (iv) are repeated so as to monitor the subject's amount of the first of the pair of mutually exclusive mutations over time. ### Claim 10 (depends on 9) The method of claim 9, wherein a decrease in amount of the first of the pair of mutually exclusive mutations indicates that the cancer is stabilizing or decreasing, or wherein an increase in amount of the first of the pair of mutually exclusive mutations indicates that the cancer is increasing. ### Claim 11 A method to monitor cell free DNA comprising: (i) obtaining a body fluid sample from a subject known to have a cancer characterized by a pair of mutually exclusive mutations specific to the cancer; (ii) isolating cell free nucleic acids from the body fluid sample obtained from the subject; (iii) measuring the level of Line 1 in the cell free nucleic acids isolated from the body fluid sample, wherein the level of Line 1 in the sample is within a range of 3,000-700,000 pg/μL; (iv) measuring the amount of the first of the pair of mutually exclusive mutations specific to the cancer in the cell free nucleic acids isolated from the body fluid sample; (v) measuring the apparent amount of the first of the pair of mutually exclusive mutations in control cell free nucleic acids isolated from body fluid samples obtained from control subjects known to have the second of the pair of mutually exclusive mutations specific to the cancer using measuring conditions substantially the same as those used to measure the amount of the first of the pair of mutually exclusive mutations in the cell free nucleic acids isolated from the body fluid sample from the subject, and (vi) indicating in a report that the subject has the first of the pair of mutually exclusive mutations when (a) the level of Line 1 in the cell free nucleic acids isolated from the body fluid sample is within the range of 3,000-700,000 pg/μL and (b) the amount of the first of the pair of mutually exclusive mutations is increased as compared to the apparent amount of the first of the pair of mutually exclusive mutations in control cell free nucleic acids isolated from body fluid samples obtained from control subjects. ### Claim 12 A method to treat cancer comprising: (i) obtaining a body fluid sample from a subject known to have a cancer characterized by a pair of mutually exclusive mutations specific to the cancer; (ii) isolating cell free nucleic acids from the body fluid sample obtained from the subject; (iii) measuring the level of Line 1 in the cell free nucleic acids isolated from the body fluid sample, wherein the level of Line 1 is within a range of 3,000-700,000 pg/μL; (iv) measuring the amount of the first of the pair of mutually exclusive mutations specific to the cancer in the cell free nucleic acids isolated from the body fluid sample; (v) measuring the apparent amount of the first of the pair of mutually exclusive mutations in control cell free nucleic acids isolated from body fluid samples obtained from control subjects known to have the second of the pair of mutually exclusive mutations specific to the cancer using measuring conditions substantially the same as those used to measure the amount of the first of the pair of mutually exclusive mutations in the cell free nucleic acids isolated from the body fluid sample from the subject; and (vi) treating the subject with an anti-cancer therapy when (a) the level of Line 1 in the cell free nucleic acids isolated from the body fluid sample is within the range of 3,000-700,000 pg/μL and (b) the amount of the first of the pair of mutually exclusive mutations is increased as compared to the apparent amount of the first of the pair of mutually exclusive mutations in control cell free nucleic acids isolated from body fluid samples obtained from control subjects. ### Claim 13 (depends on 12) The method of claim 12, wherein step (i) step (iv) are repeated so as to monitor the subject's amount of the first of the pair of mutually exclusive mutations over time. ### Claim 14 (depends on 13) The method of claim 13, wherein administration of the anti-cancer therapy is maintained when the amount of the mutation decreases over time, or wherein the anti-cancer therapy is administered at a higher dosage or is changed when the amount of the mutation increases over time. ### Claim 15 A method comprising: (i) administering an anti-cancer therapy to a subject known to have a cancer characterized by a pair of mutually exclusive mutations specific to the cancer; (ii) obtaining a body fluid sample from the subject; (iii) isolating cell free nucleic acids from the body fluid sample obtained from the subject; (iv) measuring the level of Line 1 in the cell free nucleic acids isolated from the body fluid sample, wherein the level of Line 1 in the sample is within a range of 3,000-700,000 pg/μL; (v) measuring the amount of the first of the pair of mutually exclusive mutations specific to the cancer in the cell free nucleic acids isolated from the body fluid sample; and (vi) measuring the apparent amount of the first of the pair of mutually exclusive mutations in control cell free nucleic acids isolated from body fluid samples obtained from control subjects known to have the second of the pair of mutually exclusive mutations specific to the cancer using measuring conditions substantially the same as those used to measure the amount of the first of the pair of mutually exclusive mutations in the cell free nucleic acids isolated from the body fluid sample from the subject. ### Claim 16 (depends on 7) The method claim 7, wherein the amount of the first of the pair of mutually exclusive mutations specific to the cancer is determined by: preparing at least 2 serial dilutions of the cell free nucleic acids isolated from the body fluid sample; measuring the amount of the first of the pair of mutually exclusive mutations in the at least 2 serial dilutions using digital droplet PCR; and evaluating linearity of the measured dilutions to confirm accuracy of the method. ### Claim 17 (depends on 9) The method of claim 9, wherein an increase in amount of the mutation indicates that the cancer is increasing. ### Claim 18 (depends on 9) The method of claim 9, wherein the subject's amount of the first of the pair of mutually exclusive mutations is measured: (a) in a first sample obtained from the subject before the subject received an anti-cancer therapy; and (b) in a second sample obtained from the subject after the subject received an anti-cancer therapy. ### Claim 19 (depends on 11) The method of claim 11, wherein the measuring of: (a) the first of the pair of mutually exclusive mutations specific to the cancer in the cell free nucleic acids isolated from the body fluid sample obtained from the subject and (b) the apparent amount of the first of the pair of mutually exclusive mutations in cell free nucleic acids isolated from control body fluid samples obtained from control subjects known to have the second of the pair of mutually exclusive mutations specific to the cancer is performed by microarrays, Next-generation sequencing, chemiluminescence methods, fluorescent methods, digital detection, or mass spectrometry (MALDI-TOF). ### Claim 20 A method to monitor cell free DNA comprising: (i) obtaining a body fluid sample from a subject known to have a cancer characterized by a pair of mutually exclusive mutations specific to the cancer; (ii) isolating cell free nucleic acids from the body fluid sample obtained from the subject; (iii) measuring the level of Line 1 in the cell free nucleic acids isolated from the body fluid sample, wherein the measured level of Line 1 is within a range of 3,000-700,000 pg/μL of body fluid; (iv) measuring the amount of the first of the pair of mutually exclusive mutations specific to the cancer in the cell free nucleic acids isolated from the body fluid sample; and (v) measuring the apparent amounts of the first of the pair of mutually exclusive mutations in control cell free nucleic acids isolated from body fluid samples obtained from a plurality of control subjects known to have the second of the pair of mutually exclusive mutations specific to the cancer using measuring conditions substantially the same as those used to measure the amount of the first of the pair of mutually exclusive mutations in the cell free nucleic acids isolated from the body fluid sample from the subject; and (vi) indicating a result in a report wherein: the amount measured in (iv) is greater than the highest amount measured in (v) and indicating in a report that the body fluid sample from the subject has the first of the pair of mutually exclusive mutations; or the amount measured in (iv) is less than the highest amount measured in (v) and indicating a report that the body fluid sample from the subject does not have the first of the pair of mutually exclusive mutations. ### Claim 21 (depends on 20) The method of claim 20, wherein the measuring of: the first of the pair of mutually exclusive mutations specific to the cancer in the cell free nucleic acids isolated from the body fluid sample obtained from the subject is performed by quantitative PCR, microarrays, Next-generation sequencing, chemiluminescence methods, fluorescent methods, digital detection, or mass spectrometry (MALDI-TOF). ### Claim 22 (depends on 20) The method of claim 20, wherein the cancer is lung cancer or colon cancer. ### Claim 23 (depends on 20) The method of claim 20, wherein the pair of mutually exclusive mutations is an epidermal growth factor receptor (EGFR) mutation and a Rat sarcoma (RAS) mutation, or wherein the pair of mutually exclusive mutations comprises an epidermal growth factor receptor (EGFR) mutation and a v-Ki-ras2 Kirsten rat sarcoma viral oncogene homolog (KRAS) mutation, or wherein the pair of mutually exclusive mutations comprises a v-raf murine sarcoma viral oncogene homolog B1 (BRAF) mutation and a Rat sarcoma (RAS) mutation. ### Claim 24 (depends on 23) The method of claim 23, wherein the EGFR mutation is selected from the group consisting of: L858R, T790M, L861Q, G719S, del 19 and exon 20 insertions. ### Claim 25 (depends on 20) The method of claim 20, wherein the amount of the first of the pair of mutually exclusive mutations is measured before and after administration of an anti-cancer therapy to the subject. ### Claim 26 (depends on 20) The method of claim 20, wherein step (i)-step (iv) are repeated so as to monitor the subject's amount of the first of the pair of mutually exclusive mutations over time.