# US 10,246,749 B2 · Method for diagnosing breast cancer > Agents: this data is available as JSON from the patentgrep API and MCP server — see https://patentgrep.com/llms.txt - Number: US 10,246,749 B2 - Document ID: US10246749B2 - Status: Granted - Kind: B2 - Application number: 15/075,181 - Filed: 2016-03-20 - Published: 2016-12-22 - Granted: 2019-04-02 - Earliest claimed: 2015-03-20 - Assignees: Albert-Ludwigs-Universitaet Freiburg - Applicants: Albert-Ludwigs-Universitaet Freiburg - Inventors: Elmar Stickeler; Thalia Erbes; Markus Jaeger - Web page: https://patentgrep.com/patent/US10246749B2 ## CPC - C12Q 1/6886: for cancer (immunoassay for cancer G01N33/575) - C12Q 2600/158: Expression markers - C12Q 2600/178: miRNA, siRNA or ncRNA - 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 ## Abstract The invention relates to a method of diagnosing breast cancer and to the use of biomarkers for the detection and diagnosis of breast cancer. ## Claims ### Claim 1 A method of diagnosing a subject with breast cancer, said method comprising the steps of: a. detecting and measuring the levels of the following four miR gene products: miR-21, miR125b, miR-451, and miR-155 in a cell-free test sample isolated from a whole urine sample obtained from said subject; b. comparing the respective levels of each of said four miR gene products in the subject test sample detected and measured in step (a) with its corresponding miR gene product control level in a control sample; and c. diagnosing with breast cancer the subject for whom the comparison of step (b) reveals a decrease in the respective levels of miR-21, miR-125b, and miR-451, and an increase in the level of miR-155 in the subject test sample measured in step (a) as compared to each corresponding control level, wherein said cell-free test sample is a supernatant isolated from the whole urine sample, wherein the whole urine sample has been processed to eradicate contamination with any urothelial or microbiological cell material. ### Claim 2 (depends on 1) The method of claim 1, wherein said subject test sample is contacted with (i) an oligonucleotide capable of hybridizing under high stringency conditions to a nucleic acid consisting of SEQ ID NO:1, (ii) an oligonucleotide capable of hybridizing under high stringency conditions to a nucleic acid consisting of SEQ ID NOs: 3 or 9; (iii) an oligonucleotide capable of hybridizing under high stringency conditions to a nucleic acid consisting of SEQ ID NO: 5; and (iv) an oligonucleotide capable of hybridizing under high stringency conditions to a nucleic acid consisting of SEQ ID NO: 7. ### Claim 3 (depends on 1) The method of claim 1, wherein the levels of each of said four miR gene products are measured by quantitative RT-PCR. ### Claim 4 (depends on 1) The method of claim 1, wherein the levels of each of said four miR gene products are measured by hybridization using an oligonucleotide microarray. ### Claim 5 (depends on 4) The method of claim 4, wherein said oligonucleotide microarray comprises: a. an oligonucleotide capable of hybridizing under high stringency conditions with a nucleic acid consisting of SEQ ID NO: 2; b. an oligonucleotide capable of hybridizing under high stringency conditions with a nucleic acid consisting of SEQ ID NO: 4; c. an oligonucleotide capable of hybridizing under high stringency conditions with a nucleic acid consisting of SEQ ID NO: 6; and d. an oligonucleotide capable of hybridizing under high stringency conditions with a nucleic acid consisting of SEQ ID NO: 8. ### Claim 6 (depends on 4) The method of claim 4, wherein said oligonucleotide microarray comprises: a. an oligonucleotide perfectly complementary to a nucleic acid consisting of SEQ ID NO: 2; b. an oligonucleotide perfectly complementary to a nucleic acid consisting of SEQ ID NO: 4; c. an oligonucleotide perfectly complementary to a nucleic acid consisting of SEQ ID NO: 6; and d. an oligonucleotide perfectly complementary to a nucleic acid consisting of SEQ ID NO: 8. ### Claim 7 (depends on 1) The method of claim 1, wherein the control sample is a urine sample obtained from one or more healthy, cancer-free individuals. ### Claim 8 (depends on 1) The method of claim 1, wherein the subject is a female human. ### Claim 9 (depends on 1) The method of claim 1, wherein the levels of the at least three each of said four miR gene products are measured by hybridization using an oligonucleotide microarray that comprises: a. an oligonucleotide capable of hybridizing under high stringency conditions with a nucleic acid consisting of SEQ ID NO: 2; b. an oligonucleotide capable of hybridizing under high stringency conditions with a nucleic acid consisting of SEQ ID NO: 4; c. an oligonucleotide capable of hybridizing under high stringency conditions with a nucleic acid consisting of SEQ ID NO: 6; d. an oligonucleotide capable of hybridizing under high stringency conditions with a nucleic acid consisting of SEQ ID NO: 8. ### Claim 10 (depends on 1) The method of claim 1, wherein the levels of the at-least-three each of said four miR gene products are measured by hybridization using an oligonucleotide microarray, further wherein said oligonucleotide microarray comprises at least three of the following: a. an oligonucleotide perfectly complementary to a nucleic acid consisting of SEQ ID NO: 2; b. an oligonucleotide perfectly complementary to a nucleic acid consisting of SEQ ID NO: 4; c. an oligonucleotide perfectly complementary to a nucleic acid consisting of SEQ ID NO: 6; and d. an oligonucleotide perfectly complementary to a nucleic acid consisting of SEQ ID NO: 8. ### Claim 11 (depends on 1) The method of claim 1, wherein said step c) of diagnosing said subject with breast cancer requires a decrease of 25% or more in the level of the miR-21 gene product, the miR-125b gene product, and the miR-451 gene product in the subject test sample, relative to the respective control levels of the corresponding gene products, coupled with an increase of 25% of more in the level of the miR-155 gene product in the subject test sample, relative to the control level of the miR-155 gene product. ### Claim 12 (depends on 1) The method of claim 1, wherein said step c) of diagnosing said subject with breast cancer requires that: a. the level of the miR-21 gene product in the test sample from the subject is less than 70% of the level of miR-21 gene product in the control sample; b. the level of the miR-125 b gene product in the test sample from the subject is less than 70% of the level of miR-125b gene product in the control sample; c. the level of the miR-451 gene product in the test sample from the subject is less than 60% of the level of miR-451 gene product in the control sample; d. the level of the miR-155 gene product in the test sample from the subject is more than 300% of the level of miR-155 gene product in the control sample. ### Claim 13 A method of treating breast cancer in a subject in need thereof, said method comprising the steps of: a. in a cell free test sample isolated from a whole urine sample obtained from the subject, detecting and measuring the respective levels of the following four miR gene products: miR-21, miR-125b, miR-451, and miR-155; b. comparing the levels of each of said four miR gene products in the subject test sample measured in step (a) with its corresponding miR gene product control level in a control sample; and c. administering a chemotherapeutic regimen suitable for treating breast cancer to the subject for whom the comparison of step (b) reveals a decrease in the level of the miR-21 gene product, the miR-125b gene product, and the miR-451 gene product in the subject test sample, relative to the corresponding control level for the respective gene product(s) and reveals an increase in the level of the miR-155 gene product in the subject test sample, relative to the corresponding control level for the respective gene product(s), wherein said cell-free test sample is a supernatant isolated from the whole urine sample, wherein the whole urine sample has been processed to eradicate contamination with any urothelial or microbiological cell material. ### Claim 14 (depends on 1) The method of claim 1, wherein the levels of each of said four miR gene products are measured by hybridization using an oligonucleotide microarray comprising at least four probe oligonucleotides, each of which is specific for one of said four miR gene products. ### Claim 15 (depends on 1) The method of claim 1, wherein said supernatant is isolated from said whole urine sample by means of centrifugation. ### Claim 16 (depends on 13) The method of claim 13, further comprising the step of repeating method steps (a) and (b) at regular time intervals in those subjects administered a chemotherapeutic regimen suitable for breast cancer in accordance with step (c) in order to monitor the effectiveness of said chemotherapeutic regimen, wherein an increase in the level of one or more miR gene products selected from the group consisting of miR-21, miR-125b and miR-451 and a decrease in the level of the miR-155 gene product in a test sample isolated from a urine sample obtained from said subject as compared to the initial values determined is indicative of therapeutic success.