# US 10,246,751 B2 · Method for discrimination of metaplasias from neoplastic or preneoplastic lesions > Agents: this data is available as JSON from the patentgrep API and MCP server — see https://patentgrep.com/llms.txt - Number: US 10,246,751 B2 - Document ID: US10246751B2 - Status: Granted - Kind: B2 - Application number: 15/479,961 - Filed: 2017-04-05 - Published: 2017-09-21 - Granted: 2019-04-02 - Earliest claimed: 2002-04-09 - Assignees: Ventana Medical Systems, Inc. - Applicants: Ventana Medical Systems, Inc. - Inventors: Ruediger Ridder; Magnus Von Knebel Doeberitz; Peter Martin - Web page: https://patentgrep.com/patent/US10246751B2 ## CPC - C12Q 1/6886: for cancer (immunoassay for cancer G01N33/575) - C12Q 1/708: {for papilloma} - G01N 33/5755: of the uterine cervix, uterine corpus or endometrium - C12Q 2600/112: Disease subtyping, staging or classification - C12Q 2600/158: Expression markers - G01N 2333/025: Papovaviridae, e.g. papillomavirus, polyomavirus, SV40, BK virus, JC virus - 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 - G01N: INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES (measuring or testing apparatus or processes other than immunoassay, involving enzymes or microorganisms C12M, C12Q) ## Abstract The present invention relates to a method for discrimination of p16INK4a overexpressing metaplasias from neoplastic or preneoplastic p16INK4a overexpressing lesions by determination of the level of high risk HPV encoded gene-products such as e.g. HPV E2 and/or HPV E7 molecules in biological samples in the course of cytological testing procedures. The method thus enables for reduction of false positive results in the p16INK4a based detection of anogenital lesions in cytological testing procedures. ## Claims ### Claim 1 A method for screening a uterine cervical cytology sample, comprising: (a) obtaining at least one slide having the uterine cervical cytology sample deposited thereon; (b) staining the uterine cytology sample with a labeled oligonucleotide probe that specifically binds p16INK4a mRNA and is complementary to p16INK4a mRNA in the sample by in situ hybridization, wherein the label of the oligonucleotide probe that is complementary to the p16INK4a mRNA generates a first reporter signal; (c) staining the uterine cervical cytology sample with a labelled oligonucleotide probe that specifically binds a high risk HPV (hr-HPV) gene-product and is complementary to a mRNA of the high risk HPV (hr-HPV) gene-product by in situ hybridization, wherein said high risk HPV gene-product is encoded by a gene selected from the group consisting of HPV E7 gene, HPV E2 gene, HPV E6 gene, HPV L1 gene, and HPV L2 gene, wherein the label of the oligonucleotide probe that is complementary to the mRNA of the hr-HPV gene product generates a second reporter signal; and (d) detecting in the uterine cytology sample: (d1) the simultaneous-presence of cells stained with the label generating the first reporter signal and cells stained with the label generating the second reporter signal, or (d2) the presence of cells stained with the label generating the first reporter signal and the absence of cells stained with the label generating the second reporter signal. ### Claim 2 (depends on 1) The method according to claim 1, wherein the high risk HPV gene-product is encoded by the HPV E7 gene. ### Claim 3 (depends on 1) The method according to claim 1, wherein the high risk HPV gene-product is encoded by the HPV E2 or the HPV E6 gene. ### Claim 4 (depends on 1) The method according to claim 1, wherein the high risk HPV gene-product is encoded by the HPV L1 or the HPV L2 gene. ### Claim 5 (depends on 1) The method of claim 1, wherein the staining of (b) and the staining of (c) are performed on the same slide, and wherein the first reporter signal and the second reporter signal are different. ### Claim 6 (depends on 5) The method of claim 5, wherein the agent generating the first reporter signal and the agent generating the second reporter signal are different fluorescent labels. ### Claim 7 (depends on 5) The method of claim 5, wherein the agent generating the first reporter signal and the agent generating the second reporter signal are different colored compounds. ### Claim 8 (depends on 1) The method of claim 1, wherein the staining of (b) and the staining of (c) are performed on different slides. ### Claim 9 (depends on 8) The method of claim 8, wherein the agent generating the first reporter signal and the agent generating the second reporter signal are fluorescent labels or colored compounds. ### Claim 10 (depends on 9) The method according to claim 9, wherein the colored compound that generates the first reporter signal and the second reporter signal is diaminobenzidine. ### Claim 11 A method of preparing a uterine cervical cytology sample for microscopic evaluation, said method comprising: (a) obtaining a slide having the uterine cervical cytology sample deposited thereon; (b) staining the uterine cervical cytology sample on the slide by in situ hybridization for both: (b1) p16INK4a mRNA, wherein the stain for p16INK4a mRNA is an agent that specifically binds p16INK4a mRNA and gene rates a first reporter signal; and (b2) a high risk HPV (hrHPV) mRNA encoded by a gene selected from the group consisting of HPV E7 gene, HPV E2 gene, HPV E6 gene, HPV L1 gene, and HPV L2 gene, wherein the stain for the hrHPV is an agent that specifically binds the high risk HPV mRNA and generates a second reporter signal, wherein the first reporter signal is distinguishable from the second reporter signal. ### Claim 12 (depends on 11) The method of claim 11, wherein the agent generating the first reporter signal and the agent generating the second reporter signal are different fluorescent labels or different colored compounds. ### Claim 13 (depends on 11) The method according to claim 11, wherein the high risk HPV gene-product is encoded by the HPV E7 gene. ### Claim 14 (depends on 11) The method according to claim 11, wherein the high risk HPV gene-product is encoded by the HPV E2 or the HPV E6 gene. ### Claim 15 (depends on 11) The method according to claim 11, wherein the high risk HPV gene-product is encoded by the HPV L1 or the HPV L2 gene. ### Claim 16 A method of preparing a uterine cervical cytology sample for microscopic evaluation, said method comprising: (a) obtaining at least two slides having the uterine cervical cytology sample deposited thereon; (b) staining the uterine cervical sample on at least one of the slides by in situ hybridization for p16INK4a mRNA with an agent that specifically binds p16INK4a mRNA and generates a first reporter signal; and (c) staining the uterine cervical sample on at least one of the slides by in situ hybridization for a high risk HPV (hrHPV) mRNA with an agent that specifically binds the high risk HPV mRNA and generates a reporter signal, wherein the hrHPV is encoded by a gene selected from the group consisting of HPV E7 gene, HPV E2 gene, HPV E6 gene, HPV L1 gene, and HPV L2 gene, wherein the stain for the hrHPV is an agent that generates a second reporter signal, wherein the first reporter signal is distinguishable from the second reporter signal. ### Claim 17 (depends on 16) The method of claim 16, wherein the agent generating the first reporter signal and the agent generating the second reporter signal are fluorescent labels or colored compounds. ### Claim 18 (depends on 16) The method according to claim 16, wherein the high risk HPV gene-product is encoded by the HPV E7 gene. ### Claim 19 (depends on 16) The method according to claim 16, wherein the high risk HPV gene-product is encoded by the HPV E2 or the HPV E6 gene. ### Claim 20 (depends on 16) The method according to claim 16, wherein the high risk HPV gene-product is encoded by the HPV L1 or the HPV L2 gene.