DETECTING BREAST CANCER - MAYO FOUND MEDICAL EDUCATION & RES

Title:

DETECTING BREAST CANCER

Document Type and Number:

WIPO Patent Application WO/2019/108626

Kind Code:

A1

Abstract:

Provided herein is technology for breast cancer screening and particularly, but not exclusively, to methods, compositions, and related uses for detecting the presence of breast cancer.

Inventors:

AHLQUIST DAVID A (US)
TAYLOR WILLIAM R (US)
MAHONEY DOUGLAS W (US)
YAB TRACY C (US)
KISIEL JOHN B (US)
ALLAWI HATIM T (US)
LIDGARD GRAHAM P (US)
KAISER MICHAEL W (US)

Application Number:

PCT/US2018/062809

Publication Date:

June 06, 2019

Filing Date:

November 28, 2018

Export Citation:

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Assignee:

MAYO FOUND MEDICAL EDUCATION & RES (US)
EXACT SCIENCES DEV CO LLC (US)

International Classes:

C12N15/09; C12Q1/68; C40B30/04

Domestic Patent References:

WO2017192221A1	2017-11-09
WO2016109782A2	2016-07-07

Foreign References:

US20170283886A1	2017-10-05
US20030186248A1	2003-10-02
US20150275314A1	2015-10-01
US20170292163A1	2017-10-12

Other References:

See also references of EP 3717644A4

Attorney, Agent or Firm:

GOETZ, Robert, A. (US)

Download PDF:

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Claims:

CLAIMS

WE CLAIM:

1. A method for characterizing a biological sample comprising:

(a) measuring a methylation level of a CpG site for one or more genes selected from BHLHE23 C, CALN1 A, CD1D, HOXA7_A, LOC100132891, MAX. chr 1.8277479- 8277527, MAX.chrl5.968890l3-96889l28, NACAD, ATP6V1B1, BANK1, Cl7orf64, DLX4, EMX1 A, FOXP4, GP5, ITPRIPL1, LMX1B A, MAX.chrl 1.14926602-14927148, MAX. chr5.42994866-42994936, MAX.chr8.124173030-124173395, MPZ, PRKCB, STX16 B UBTF, LOC100132891, ITPRIPL1, ABLIM1, MAX.chrl9.46379903-46380l97, ZSCAN12, BHLHE23 D, CXCL12, KCNK9, OTX1, RIC3, SCRT2 B,

MAX. chrl 7.73073682-73073814, CDH4_E, HNF1B B, TRH A, MAX. chr20.1784209- 1784461, MAX. chr5.145725410-145725459, MAX.chr5.77268672-77268725, BEST4, and DSCR6 in a biological sample of a human individual through

treating genomic DNA in the biological sample with bisulfite; amplifying the bi sulfite-treated genomic DNA using a set of primers for the selected one or more genes; and

determining the methylation level of the CpG site by methylation-specific PCR, quantitative methylation-specific PCR, methylation-sensitive DNA restriction enzyme analysis, quantitative bisulfite pyrosequencing, or bisulfite genomic sequencing PCR;

(b) comparing the methylation level to a methylation level of a corresponding set of genes in control samples without breast cancer; and

(c) determining that the individual has BRCA1 cancer when the methylation level measured in the one or more genes is higher than the methylation level measured in the respective control samples.

2. The method of claim 1 wherein the set of primers for the selected one or more genes is selected from the group consisting of:

for BHLHE23 C a set of primers consisting of SEQ ID NOS: 19 and 20,

for BHLHE23 D a set of primers consisting of SEQ ID NOS: 21 and 22,

for CALNl_A a set of primers consisting of SEQ ID NOS: 29 and 30, for CD1D a set of primers consisting of SEQ ID NOS: 33 and 34,

for HOXA7_A a set of primers consisting of SEQ ID NOS: 89 and 90,

for LOC100132891 a set of primers selected from the group consisting of SEQ ID NOS: 117 and 118 or SEQ ID NOS: 119 and 120,

for MAX. chr 1.8277479-8277527 a set of primers consisting of SEQ ID NOS: 127 and 128, for MAX. chrl5.96889013-96889128 a set of primers consisting of SEQ ID NOS: 139 and 140,

for NACAD a set of primers consisting of SEQ ID NOS: 179 and 180,

for ATP6V1B1 a set of primers consisting of SEQ ID NOS: 13 and 14,

for BANK1 a set of primers consisting of SEQ ID NOS: 15 and 16,

for Cl7orf64 a set of primers consisting of SEQ ID NOS: 25 and 26,

for DLX4 a set of primers consisting of SEQ ID NOS: 51 and 52,

for EMXl_A a set of primers consisting of SEQ ID NOS: 61 and 62,

for FOXP4 a set of primers consisting of SEQ ID NOS: 73 and 74,

for GP5 a set of primers consisting of SEQ ID NOS: 77 and 78,

for LMX1B A a set of primers consisting of SEQ ID NOS: 115 and 116,

for MAX.chrl 1.14926602-14927148 a set of primers consisting of SEQ ID NOS: 129 and

130,

for MAX. chr5.42994866-42994936 a set of primers consisting of SEQ ID NOS: 159 and 160, for MAX. chr8.124173030-124173395 a set of primers consisting of SEQ ID NOS: 165 and 166,

for MPZ a set of primers consisting of SEQ ID NOS: 175 and 176,

for PRKCB a set of primers consisting of SEQ ID NOS: 201 and 202,

for STXl6_B a set of primers consisting of SEQ ID NOS: 233 and 234 or SEQ ID NOS: 235 and 236,

for UBTF a set of primers consisting of SEQ ID NOS: 249 and 250,

for LOC100132891 a set of primers selected from the group consisting of SEQ ID NOS: 117 and 118 or SEQ ID NOS: 119 and 120,

for ITPRIPL1 a set of primers selected from the group consisting of from SEQ ID NOS: 97 and 98 and SEQ ID NOS: 99 and 100,

for ABLIM1 a set of primers consisting of SEQ ID NOS: 3 and 4,

for MAX. chrl9.46379903-46380197 a set of primers selected from the group consisting of SEQ ID NOS: 145 and 146 and SEQ ID NOS: 147 and 148, for ZSCAN12 a set of primers consisting of SEQ ID NOS: 251 and 252,

for CXCL12 a set of primers consisting of SEQ ID NOS: 47 and 48,

for KCNK9 a set of primers consisting of SEQ ID NOS: 105 and 106,

for OTX1 a set of primers consisting of SEQ ID NOS: 189 and 190,

for RIC3 a set of primers consisting of SEQ ID NOS: 209 and 210,

for SCRT2 B a set of primers consisting of SEQ ID NOS: 211 and 212,

for MAX. chrl7.73073682-73073814 a set of primers consisting of SEQ ID NOS: 141 and

142,

for CDH4 E a set of primers selected from the group consisting of SEQ ID NOS: 35 and 36, for HNF1B B a set of primers consisting of SEQ ID NOS: 85 and 86,

for TRH A a set of primers consisting of SEQ ID NOS: 245 and 246,

for MAX. chr20.1784209-1784461 a set of primers selected from the group consisting of SEQ

ID NOS: 151 and 152 and SEQ ID NOS: 153 and 154,

for MAX. chr5.145725410-145725459 a set of primers consisting of SEQ ID NOS: 157 and 158,

for MAX. chr5.77268672-77268725 a set of primers consisting of SEQ ID NOS: 161 and 162, for BEST4 a set of primers consisting of SEQ ID NOS: 17 and 18, and

for DSCR6 a set of primers consisting of SEQ ID NOS: 57 and 58.

3. The method of claim 1, wherein the biological sample is a plasma sample, a blood sample, or a tissue sample (e.g., breast tissue).

4. The method of claim 1, wherein the one or more genes is described by the genomic coordinates shown in Table 2.

5. The method of claim 1, wherein said CpG site is present in a coding region or a

regulatory region.

6. The method of claim 1, wherein said measuring the methylation level a CpG site for one or more genes comprises a determination selected from the group consisting of determining the methylation score of said CpG site and determining the methylation frequency of said CpG site.

7. A method for characterizing a biological sample comprising:

(a) measuring a methylation level of a CpG site for one or more genes selected from MAX. chrl5.968890l3-96889l28, ATP6V1B1, Cl7orf64, ITPRIPL1,

MAX. chrl 1.14926602-14927148, MAX. chr5.42994866-42994936, LOC 100132891, ITPRIPL1, ABLIM1, MAX.chrl9.46379903-46380l97, COL23A1, LAYN, OTX1, TRH A, MAX.chr5.145725410-145725459, and MAX. chrl 1.68622869-68622968 in a biological sample of a human individual through

treating genomic DNA in the biological sample with bisulfite; amplifying the bi sulfite-treated genomic DNA using a set of primers for the selected one or more genes; and

determining the methylation level of the CpG site by methylation-specific PCR, quantitative methylation-specific PCR, methylation-sensitive DNA restriction enzyme analysis, quantitative bisulfite pyrosequencing, or bisulfite genomic sequencing PCR;

(b) comparing the methylation level to a methylation level of a corresponding set of genes in control samples without breast cancer; and

(c) determining that the individual has BRC A2 cancer when the methylation level measured in the one or more genes is higher than the methylation level measured in the respective_control samples.

8. The method of claim 7 wherein the set of primers for the selected one or more genes is selected from the group consisting of:

for MAX. chrl5.96889013-96889128 a set of primers consisting of SEQ ID NOS: 139 and 140,

for ATP6V1B1 a set of primers consisting of SEQ ID NOS: 13 and 14,

for Cl7orf64 a set of primers consisting of SEQ ID NOS: 25 and 26

for MAX. chrl 1.14926602-14927148 a set of primers consisting of SEQ ID NOS: 129 and

130,

for MAX. chr5.42994866-42994936 a set of primers consisting of SEQ ID NOS: 159 and 160, for LOC100132891 a set of primers selected from the group consisting of SEQ ID NOS: 117 and 118 or SEQ ID NOS: 119 and 120,

for ITPRIPL1 a set of primers consisting of selected from the group consisting of SEQ ID NOS: 97 and 98 and SEQ ID NOS: 99 and 100, for ABLIM1 a set of primers consisting of SEQ ID NOS: 3 and 4

for MAX. chrl9.46379903-46380197 a set of primers selected from the group consisting of

SEQ ID NOS: 145 and 146 and SEQ ID NOS: 147 and 148,

for COL23A1 a set of primers consisting of SEQ ID NOS: 45 and 46,

for LAYN a set of primers consisting of SEQ ID NOS: 111 and 112,

for OTX1 a set of primers consisting of SEQ ID NOS: 189 and 190,

for TRH A a set of primers consisting of SEQ ID NOS: 245 and 246,

for MAX. chr5.145725410-145725459 a set of primers consisting of SEQ ID NOS: 157 and

158, and

for MAX. chr 11.68622869-68622968 a set of primers consisting of SEQ ID NOS: 131 and 132.

9. The method of claim 7, wherein the biological sample is a plasma sample, a blood sample, or a tissue sample (e.g., breast tissue).

10. The method of claim 7, wherein the one or more genes is described by the genomic coordinates shown in Table 2.

11. The method of claim 7, wherein said CpG site is present in a coding region or a

regulatory region.

12. The method of claim 7, wherein said measuring the methylation level a CpG site for one or more genes comprises a determination selected from the group consisting of determining the methylation score of said CpG site and determining the methylation frequency of said CpG site.

13. A method for characterizing a biological sample comprising:

(a) measuring a methylation level of a CpG site for one or more genes selected from ATP6V1B1, MAX. chrl l.14926602-14927148, PRKCB, TRH A, MPZ, GP5, TRIM67, MAX. chrl 2.4273906-4274012, CALN1 A, MAX.chrl2.4273906-42740l2,

MAX. chr5.42994866-42994936, SCRT2_B, MAX.chr5.145725410-145725459,

BHLHE23 D, MAX.chr5.77268672-77268725, EMX1 A, DSCR6, and DLX4 in a biological sample of a human individual through treating genomic DNA in the biological sample with bisulfite; amplifying the bi sulfite-treated genomic DNA using a set of primers for the selected one or more genes; and

determining the methylation level of the CpG site by methylation-specific PCR, quantitative methylation-specific PCR, methylation-sensitive DNA restriction enzyme analysis, quantitative bisulfite pyrosequencing, or bisulfite genomic sequencing PCR;

(b) comparing the methylation level to a methylation level of a corresponding set of genes in control samples without breast cancer; and

(c) determining that the individual has triple negative cancer when the

methylation level measured in the one or more genes is higher than the methylation level measured in the respective_control samples.

14. The method of claim 13 wherein the set of primers for the selected one or more genes is selected from the group consisting of:

for ATP6V1B1 a set of primers consisting of SEQ ID NOS: 13 and 14,

for MAX.chrl 1.14926602-14927148 a set of primers consisting of SEQ ID NOS: 129 and

130,

for PRKCB a set of primers consisting of SEQ ID NOS: 201 and 202,

for TRH A a set of primers consisting of SEQ ID NOS: 245 and 246,

for MPZ a set of primers consisting of SEQ ID NOS: 175 and 176,

for GP5 a set of primers consisting of SEQ ID NOS: 77 and 78,

for TRIM67 a set of primers consisting of SEQ ID NOS: 247 and 248,

for MAX. chrl2.4273906-4274012 a set of primers selected from the group consisting of SEQ

ID NOS: 133 and 134 and SEQ ID NOS: 135 and 136,

for CALNl_A a set of primers consisting of SEQ ID NOS: 29 and 30,

for MAX. chr5.42994866-42994936 a set of primers consisting of SEQ ID NOS: 159 and 160, for SCRT2 B a set of primers consisting of SEQ ID NOS: 211 and 212,

for MAX. chr5.145725410-145725459 a set of primers consisting of SEQ ID NOS: 157 and

158,

for BHLHE23 D a set of primers consisting of SEQ ID NOS: 21 and 22,

for MAX. chr5.77268672-77268725 a set of primers consisting of SEQ ID NOS: 161 and 162, for EMXl_A a set of primers consisting of SEQ ID NOS: 61 and 62, for DSCR6 a set of primers consisting of SEQ ID NOS: 57 and 58, and

for DLX4 a set of primers consisting of SEQ ID NOS: 51 and 52.

15. The method of claim 13, wherein the biological sample is a plasma sample, a blood sample, or a tissue sample (e.g., breast tissue).

16. The method of claim 13, wherein the one or more genes is described by the genomic coordinates shown in Table 2.

17. The method of claim 13, wherein said CpG site is present in a coding region or a regulatory region.

18. The method of claim 13, wherein said measuring the methylation level a CpG site for one or more genes comprises a determination selected from the group consisting of determining the methylation score of said CpG site and determining the methylation frequency of said CpG site.

19. A method for characterizing a biological sample comprising:

(a) measuring a methylation level of a CpG site for one or more genes selected from ATP6V1B1, LMX1B A, BANK1, OTX1, MAX.chrl 1.14926602-14927148, UBTF, PRKCB, TRH A, MPZ, GP5, DNM3 A, TRIM67, PLXNC1 A, MAX.chrl2.4273906- 4274012, CALN1 A, ITPRIPL1, MAX.chrl2.4273906-42740l2, GYPC_B,

M AX. chr5.42994866-42994936, OSR2_A, SCRT2_B, MAX.chr5.145725410-145725459, MAX. chrl 1.68622869-68622968, MAX.chr8.124173030-124173395, MAX.chr20.1784209- 1784461, LOC100132891, BHLHE23 C, ALOX5, MAX.chrl9.46379903-46380l97, ODC1, CHST2 A, M AX. chr5.77268672-77268725, Cl7orf64, EMX1 A, CHST2_B, DSCR6, ITPRIPL1, IGF2BP3 B, DLX4, ABLIM1, BHLHE23 D, ZSCAN12, GRASP, and

Cl0orfl25 in a biological sample of a human individual through

treating genomic DNA in the biological sample with bisulfite; amplifying the bisulfite-treated genomic DNA using a set of primers for the selected one or more genes; and

determining the methylation level of the CpG site by methylation-specific PCR, quantitative methylation-specific PCR, methylation-sensitive DNA restriction enzyme analysis, quantitative bisulfite pyrosequencing, or bisulfite genomic sequencing PCR;

(b) comparing the methylation level to a methylation level of a corresponding set of genes in control samples without breast cancer; and

(c) determining that the individual has HER2⁺ cancer when the methylation level measured in the one or more genes is higher than the methylation level measured in the respective_control samples.

20. The method of claim 19 wherein the set of primers for the selected one or more genes is selected from the group consisting of:

for ATP6V1B1 a set of primers consisting of SEQ ID NOS: 13 and 14,

for LMX1B A a set of primers consisting of SEQ ID NOS: 115 and 116,

for BANK1 a set of primers consisting of SEQ ID NOS: 15 and 16,

for OTX1 a set of primers consisting of SEQ ID NOS: 189 and 190,

for MAX.chrl 1.14926602-14927148 a set of primers consisting of SEQ ID NOS: 129 and

130,

for UBTF a set of primers consisting of SEQ ID NOS: 249 and 250,

for PRKCB a set of primers consisting of SEQ ID NOS: 201 and 202,

for TRH A a set of primers consisting of SEQ ID NOS: 245 and 246,

for MPZ a set of primers consisting of SEQ ID NOS: 175 and 176,

for GP5 a set of primers consisting of SEQ ID NOS: 77 and 78,

for DNM3_A a set of primers consisting of SEQ ID NOS: 55 and 56,

for TRIM67 a set of primers consisting of SEQ ID NOS: 247 and 248,

for PLXNCl_A a set of primers consisting of SEQ ID NOS: 193 and 194,

for MAX. chrl2.4273906-4274012 a set of primers selected from the group consisting of SEQ

ID NOS: 133 and 134 and SEQ ID NOS: 135 and 136,

for CALNl_A a set of primers consisting of SEQ ID NOS: 29 and 30,

for ITPRIPL1 a set of primers consisting of selected from the group consisting of SEQ ID

NOS: 97 and 98 and SEQ ID NOS: 99 and 100,

for GYPC B a set of primers consisting of SEQ ID NOS: 81 and 82,

for MAX. chr5.42994866-42994936 a set of primers consisting of SEQ ID NOS: 159 and 160, for OSR2_A a set of primers consisting of SEQ ID NOS: 187 and 188,

for SCRT2 B a set of primers consisting of SEQ ID NOS: 211 and 212, for MAX. chr5.145725410-145725459 a set of primers consisting of SEQ ID NOS: 157 and 158,

for MAX. chr 11.68622869-68622968 a set of primers consisting of SEQ ID NOS: 131 and 132,

for MAX. chr8.124173030-124173395 a set of primers consisting of SEQ ID NOS: 165 and 166,

for MAX. chr20.1784209-1784461 a set of primers selected from the group consisting of SEQ ID NOS: 151 and 152 and SEQ ID NOS: 153 and 154,

for LOC100132891 a set of primers selected from the group consisting of SEQ ID NOS: 117 and 118 or SEQ ID NOS: 119 and 120,

for BHLHE23 C a set of primers consisting of SEQ ID NOS: 19 and 20,

for ALOX5 a set of primers consisting of SEQ ID NOS: 9 and 10,

for MAX. chrl9.46379903-46380197 a set of primers selected from the group consisting of SEQ ID NOS: 145 and 146 and SEQ ID NOS: 147 and 148,

for ODCl a set of primers consisting of SEQ ID NOS: 181 and 182, SEQ ID NOS: 183 and 184, and SEQ ID NOS: 185 and 186,

for CHST2_A a set of primers consisting of SEQ ID NOS: 37 and 38,

for CHST2 B a set of primers consisting of and SEQ ID NOS: 39 and 40,

for MAX. chr5.77268672-77268725 a set of primers consisting of SEQ ID NOS: 161 and 162, for Cl7orf64 a set of primers consisting of SEQ ID NOS: 25 and 26,

for EMXl_A a set of primers consisting of SEQ ID NOS: 61 and 62,

for DSCR6 a set of primers consisting of SEQ ID NOS: 57 and 58,

for ITPRIPL1 a set of primers consisting of selected from the group consisting of SEQ ID

NOS: 97 and 98 and SEQ ID NOS: 99 and 100,

for IGF2BP3 B a set of primers consisting of SEQ ID NOS: 93 and 94,

for DLX4 a set of primers consisting of SEQ ID NOS: 51 and 52,

for ABLIM1 a set of primers consisting of SEQ ID NOS: 3 and 4,

for BHLHE23 D a set of primers consisting of SEQ ID NOS: 21 and 22,

for ZSCAN12 a set of primers consisting of SEQ ID NOS: 251 and 252,

for GRASP a set of primers consisting of SEQ ID NOS: 253 and 254, and

for Cl0orfl25 a set of primers consisting of SEQ ID NOS: 23 and 24.

21. The method of claim 19, wherein the biological sample is a plasma sample, a blood sample, or a tissue sample (e.g., breast tissue).

22. The method of claim 19, wherein the one or more genes is described by the genomic coordinates shown in Table 2.

23. The method of claim 19, wherein said CpG site is present in a coding region or a regulatory region.

24. The method of claim 19, wherein said measuring the methylation level a CpG site for one or more genes comprises a determination selected from the group consisting of determining the methylation score of said CpG site and determining the methylation frequency of said CpG site.

25. A method for characterizing a biological sample comprising:

(a) measuring a methylation level of a CpG site for one or more genes selected from ATP6V1B1, LMX1B A, BANK1, OTX1, ST8SIA4, MAX. chrl l.14926602-14927148, UBTF, PRKCB, TRH A, MPZ, DNM3 A, TRIM67, PLXNC1 A, MAX.chrl2.4273906- 4274012, CALN1 A, ITPRIPL1, MAX.chrl2.4273906-42740l2, GYPC_B,

M AX. chr5.42994866-42994936, OSR2_A, SCRT2_B, MAX.chr5.145725410-145725459, MAX. chrl 1.68622869-68622968, MAX.chr8.124173030-124173395, MAX.chr20.1784209- 1784461, LOC100132891, BHLHE23_D, ALOX5, MAX.chrl9.46379903-46380l97,

ODC1, CHST2 A, MAX.chr5.77268672-77268725, Cl7orf64, EMX1 A, CHST2_B, ITPRIPL1, IGF2BP3 B, CDH4 E, ABLIM1, SLC30A10, Cl0orfl25 in a biological sample of a human individual through

treating genomic DNA in the biological sample with bisulfite; amplifying the bi sulfite-treated genomic DNA using a set of primers for the selected one or more genes; and

determining the methylation level of the CpG site by methylation-specific PCR, quantitative methylation-specific PCR, methylation-sensitive DNA restriction enzyme analysis, quantitative bisulfite pyrosequencing, or bisulfite genomic sequencing PCR; (b) comparing the methylation level to a methylation level of a corresponding set of genes in control samples without breast cancer; and

(c) determining that the individual has Luminal A cancer when the methylation level measured in the one or more genes is higher than the methylation level measured in the respective_control samples.

26. The method of claim 25 wherein the set of primers for the selected one or more genes is selected from the group consisting of:

for ATP6V1B1 a set of primers consisting of SEQ ID NOS: 13 and 14,

for LMX1B A a set of primers consisting of SEQ ID NOS: 115 and 116,

for BANK1 a set of primers consisting of SEQ ID NOS: 15 and 16,

for OTX1 a set of primers consisting of SEQ ID NOS: 189 and 190,

for ST8SIA4 a set of primers consisting of SEQ ID NOS: 227 and 228,