# US 12,760,372 B2 · Methods and kits for determining the risk of breast cancer recurrence

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- Number: US 12,760,372 B2
- Document ID: US12760372B2
- Status: Granted
- Kind: B2
- Application number: 17/907,601
- Filed: 2021-05-07
- Published: 2023-05-11
- Granted: 2026-10-13
- Earliest claimed: 2020-05-08
- Assignees: Istituto Europeo di Oncologia S.r.l.
- Applicants: Istituto Europeo di Oncologia S.r.l.; Universita degli Studi di Milano
- Inventors: Pier Paolo Di Fiore; Salvatore Pece
- Web page: https://patentgrep.com/patent/US12760372B2

## CPC

- C12Q 1/6886: for cancer (immunoassay for cancer G01N33/575)
- G16H 50/30: for calculating health indices; for individual health risk assessment
- C12Q 2600/118: Prognosis of disease development
- C12Q 2600/158: Expression 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
- G16H: HEALTHCARE INFORMATICS, i.e. INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR THE HANDLING OR PROCESSING OF MEDICAL OR HEALTHCARE DATA

## Abstract

The present invention relates to methods and kits for the evaluation of risk and treatment of distant recurrence of breast cancer in a subject. The methods and kits can comprise combining the measured expression levels of a subset of genes selected from a 20-gene signature with tumor size and nodal status to determine the risk of distant recurrence.

## Claims

### Claim 1

A method comprising:
(a) determining, in a sample from a subject having at least one ER+/HER2− breast cancer tumor, mRNA expression levels of the following genes: H2AFZ, CDK1, EXOSC4, PHLDA2, APOBEC3B, EIF4EBP1, SFN, PHB, EPB41L5, RACGAP1, MRPS23, TOP2A, H2AFJ, NOL3, MIEN1, CENPW, LY6E, ALYREF, MMP1, and NDUFB10;
(b) determining the tumor size (pT) of the at least one ER+/HER2− breast cancer tumor;
(c) determining the nodal status (pN) of the at least one ER+/HER2− breast cancer tumor; and
(d) calculating a risk score for ER+/HER2− breast cancer recurrence based upon the mRNA expression levels of H2AFZ, CDK1, EXOSC4, PHLDA2, APOBEC3B, EIF4EBP1, SFN, PHB, EPB41L5, RACGAP1, MRPS23, TOP2A, H2AFJ, NOL3, MIEN1, CENPW, LY6E, ALYREF, MMP1, and NDUFB10, the pT and the pN; and
(e) administering at least one treatment to the subject based on the risk score, wherein the treatment comprises surgery, radiation, anthracycline agents, alkylating agents, nucleoside analogs, platinum agents, vinca agents, anti-estrogen drugs, aromatase inhibitors, ovarian suppression agents, endocrine/hormonal agents, bisphophonate therapy agents, cyclophosphamide, fluorouracil (or 5-fluorouracil or 5-FU), methotrexate, thiotepa, carboplatin, cisplatin, gemcitabine, anthracycline, taxanes, paclitaxel, protein-bound paclitaxel, doxorubicin, docetaxel, vinorelbine, tamoxifen, raloxifene, toremifene, fulvestrant, irinotecan, ixabepilone, temozolmide, topotecan, vincristine, vinblastine, eribulin, mutamycin, capecitabine, capecitabine, anastrozole, exemestane, letrozole, leuprolide, abarelix, buserlin, goserelin, megestrol acetate, risedronate, pamidronate, ibandronate, alendronate, denosumab, zoledronate, trastuzumab, tykerb or bevacizumab, or combinations thereof,
wherein the risk score is calculated according to the following formula:
Risk
⁢
Score
=
(
0
.
0
⁢
3
⁢
6
⁢
0
⁢
4
)
×
(
∑
i
β
i
×
C
⁢
q
normalized
)
+
p
⁢
T
+
p
⁢
N
5
.
6
⁢
4
⁢
0
⁢
3
⁢
2
wherein
i is the summation index for the genes for which the mRNA expression level was measured;
β is the ridge penalized Cox model coefficient for each gene as set forth below:
H2AFZ-0.03833591325196550, CDK1-0.06132455806571770, EXOSC4-0.02105976326055420, PHLDA2-0.06295739658169650, APOBEC3B 0.02341881674020150, EIF4EBP1-0.13911217901125500, SFN 0.05788269046891110, PHB-0.03538557745953510, EPB41L5-0.04675539403890050, RACGAP1-0.05097505893853430, MRPS23-0.14201022110072700, TOP2A-0.11290078348786600, H2AFJ-0.04975471358452700, NOL3-0.04193802459521500, MIEN1 0.01133668644106850, CENPW-0.03717918353187610, LY6E-0.02829256296234230, ALYREF-0.09541915699494330, MMP1-0.00911370427072023, NDUFB10 0.00626166874136819;
and Cqnormalized is the normalized average quantification cycle (Cq) for each of the genes for which the mRNA expression level was measured;
wherein
pT is 0.45542 when the tumor size of the at least one ER+/HER2− breast cancer tumor is determined to be pT1c; or
pT is 0.96572 when the tumor size of the ER+/HER2− breast cancer tumor is determined to be any of pT2, pT3 and pT4; and
wherein
pN is 0.46293 when the nodal status of the at least one ER+/HER2− breast cancer tumor is determined to be pN1 or
pN is 1.24922 when the nodal status of the at least one ER+/HER2− breast cancer tumor is determined to be pN2.

### Claim 2 (depends on 1)

The method of claim 1, wherein Cqnormalized is normalized to the expression of at least two reference genes.

### Claim 3 (depends on 2)

The method of claim 2, wherein the at least two reference genes are each
(a) housekeeping genes; or
(b) selected from the group consisting of GAPDH, GUSB, HPRTI, and TBP.

### Claim 4 (depends on 1)

The method of claim 1, wherein Cqnormalized is calculated according to the following formula:
Cq
normalized
=
AVG
⁢
Cq
-
SF
wherein SF is the difference between the AVG Cq value of the at least two reference genes for each subject and a constant reference value K.

### Claim 5 (depends on 4)

The method of claim 4, wherein (a) K is the mean of the AVG Cq of the at least two reference genes calculated across a plurality of training samples or (b) wherein K=25.012586069.

### Claim 6 (depends on 1)

The method of claim 1, wherein the mRNA expression levels are determined using one or more techniques selected from the group consisting of analysis of single strand conformation polymorphism, capillary electrophoresis, denaturing high performance liquid chromatography, digital molecular barcoding technology, direct sequencing, DNA mismatch-binding protein assays, dynamic allele-specific hybridization, Fluorescent in situ hybridization (FISH), high-density oligonucleotide SNP arrays, high-resolution melting analysis, microarray, next generation sequencing (NGS), Northern blot analysis, nuclease protection analysis, oligonucleotide ligase assays, polymerase chain reaction (PCR), primer extension assays, Quantigene analysis, quantitative nuclease-protection assay (qNPA), reporter gene detection, restriction fragment length polymorphism (RFLP) assays, reverse transcription and real-time quantitative polymerase chain reaction (RT-qPCR), reverse transcription-polymerase chain reaction (RT-PCR), RNA sequencing (RNA-seq), Serial analysis of gene expression (SAGE), Single Molecule Real Time (SMRT) DNA sequencing technology, SNPLex, Southern blot analysis, Sybr Green chemistry, TaqMan-based assays, temperature gradient gel electrophoresis (TGGE), and Tiling array.

### Claim 7

A method for stratifying a subject having at least one ER+/HER2− breast cancer tumor into a low or high risk group of ER+/HER2− breast cancer recurrence, the method comprising:
(a) determining, in a sample from the subject, mRNA expression levels of the following genes: H2AFZ, CDK1, EXOSC4, PHLDA2, APOBEC3B, EIF4EBP1, SFN, PHB, EPB41L5, RACGAP1, MRPS23, TOP2A, H2AFJ, NOL3, MIEN1, CENPW, LY6E, ALYREF, MMP1, and NDUFB10;
(b) determining the tumor size (pT) of the at least one ER+/HER2− breast cancer tumor;
(c) determining the nodal status (pN) of the at least one ER+/HER2− breast cancer tumor;
(d) calculating a risk score based upon the mRNA expression levels of H2AFZ, CDK1, EXOSC4, PHLDA2, APOBEC3B, EIF4EBP1, SFN, PHB, EPB41L5, RACGAP1, MRPS23, TOP2A, H2AFJ, NOL3, MIEN1, CENPW, LY6E, ALYREF, MMP1, and NDUFB10, the pT and the pN;
(e) stratifying the subject based upon the calculated risk score; and
(f) administering at least one treatment to the subject based on the risk score, wherein the treatment comprises surgery, radiation, anthracycline agents, alkylating agents, nucleoside analogs, platinum agents, vinca agents, anti-estrogen drugs, aromatase inhibitors, ovarian suppression agents, endocrine/hormonal agents, bisphophonate therapy agents, cyclophosphamide, fluorouracil (or 5-fluorouracil or 5-FU), methotrexate, thiotepa, carboplatin, cisplatin, gemcitabine, anthracycline, taxanes, paclitaxel, protein-bound paclitaxel, doxorubicin, docetaxel, vinorelbine, tamoxifen, raloxifene, toremifene, fulvestrant, irinotecan, ixabepilone, temozolmide, topotecan, vincristine, vinblastine, eribulin, mutamycin, capecitabine, capecitabine, anastrozole, exemestane, letrozole, leuprolide, abarelix, buserlin, goserelin, megestrol acetate, risedronate, pamidronate, ibandronate, alendronate, denosumab, zoledronate, trastuzumab, tykerb or bevacizumab, or combinations thereof,
wherein the risk score is calculated according to the following formula:
Risk
⁢
Score
=
(
0
.
0
⁢
3
⁢
6
⁢
0
⁢
4
)
×
(
∑
i
⁢
β
i
×
C
⁢
q
normalized
)
+
pT
+
pN
5
.
6
⁢
4
⁢
0
⁢
3
⁢
2
wherein
i is the summation index for the genes for which the mRNA expression level was measured;
β is the ridge penalized Cox model coefficient for each gene as set forth below:
H2AFZ-0.03833591325196550, CDK1-0.06132455806571770, EXOSC4-0.02105976326055420, PHLDA2-0.06295739658169650, APOBEC3B 0.02341881674020150, EIF4EBP1-0.13911217901125500, SEN 0.05788269046891110, PHB-0.03538557745953510, EPB41L5-0.04675539403890050, RACGAP1-0.05097505893853430, MRPS23-0.14201022110072700, TOP2A-0.11290078348786600, H2AFJ-0.04975471358452700, NOL3-0.04193802459521500, MIEN1 0.01133668644106850, CENPW-0.03717918353187610, LY6E-0.02829256296234230, ALYREF-0.09541915699494330, MMP1-0.00911370427072023, NDUFB10 0.00626166874136819;
and Cqnormalized is the normalized average quantification cycle (Cq) for each of the genes for which the mRNA expression level was measured;
wherein
pT is 0.45542 when the tumor size of the at least one ER+/HER2− breast cancer tumor is determined to be pT1c; or
pT is 0.96572 when the tumor size of the ER+/HER2− breast cancer tumor is determined to be any of pT2, pT3 and pT4; and
wherein
pN is 0.46293 when the nodal status of the at least one ER+/HER2− breast cancer tumor is determined to be pN1 or
pN is 1.24922 when the nodal status of the at least one ER+/HER2− breast cancer tumor is determined to be pN2.

### Claim 8 (depends on 7)

The method of claim 7, wherein stratifying the subject based upon the calculated risk score comprises (a) stratifying the subject as having a low risk for ER+/HER2− breast cancer recurrence when the risk score is less than a predetermined cutoff value and/or (b) stratifying the subject as having a high risk for ER+/HER2− breast cancer recurrence when the risk score is greater than a predetermined cutoff value.

### Claim 9 (depends on 7)

The method of claim 7, wherein Cqnormalized is normalized to the expression of at least two reference genes.

### Claim 10 (depends on 9)

The method of claim 9, wherein the at least two reference genes are
(a) housekeeping genes or
(b) selected from the group consisting of GAPDH, GUSB, HPRT1, and TBP.

### Claim 11 (depends on 7)

The method of claim 7, wherein Cqnormalized is calculated according to the following formula:
Cq
normalized
=
AVG
⁢
Cq
-
SF
wherein SF is the difference between the AVG Cq value of the at least two reference genes for each subject and a constant reference value K.

### Claim 12 (depends on 11)

The method of claim 11, wherein (a) K is the mean of the AVG Cq of the at least two reference genes calculated across a plurality of training samples or (b) wherein K=25.012586069.

### Claim 13 (depends on 7)

The method of claim 7, wherein the mRNA expression levels are determined using one or more techniques selected from the group consisting of analysis of single strand conformation polymorphism, capillary electrophoresis, denaturing high performance liquid chromatography, digital molecular barcoding technology, direct sequencing, DNA mismatch-binding protein assays, dynamic allele-specific hybridization, Fluorescent in situ hybridization (FISH), high-density oligonucleotide SNP arrays, high-resolution melting analysis, microarray, next generation sequencing (NGS), Heliscope instrument, Northern blot analysis, nuclease protection analysis, oligonucleotide ligase assays, polymerase chain reaction (PCR), primer extension assays, Quantigene analysis, quantitative nuclease-protection assay (qNPA), reporter gene detection, restriction fragment length polymorphism (RFLP) assays, reverse transcription and real-time quantitative polymerase chain reaction (RT-qPCR), reverse transcription-polymerase chain reaction (RT-PCR), RNA sequencing (RNA-seq), Serial analysis of gene expression (SAGE), Single Molecule Real Time (SMRT) DNA sequencing technology, SNPLex, Southern blot analysis, Sybr Green chemistry, TaqMan-based assays, temperature gradient gel electrophoresis (TGGE), and Tiling array.

### Claim 14

A method for stratifying a subject having at least one ER+/HER2− breast cancer tumor into a low, intermediate, or high risk group of ER+/HER2− breast cancer recurrence, the method comprising:
(a) determining, in a sample from the subject, mRNA expression levels of the following genes: H2AFZ, CDK1, EXOSC4, PHLDA2, APOBEC3B, EIF4EBP1, SFN, PHB, EPB41L5, RACGAP1, MRPS23, TOP2A, H2AFJ, NOL3, MIEN1, CENPW, LY6E, ALYREF, MMP1, and NDUFB10;
(b) determining the tumor size (pT) of the at least one ER+/HER2− breast cancer tumor;
(c) determining the nodal status (pN) of the at least one ER+/HER2− breast cancer tumor;
(d) calculating a risk score based upon the mRNA expression levels of H2AFZ, CDK1, EXOSC4, PHLDA2, APOBEC3B, EIF4EBP1, SFN, PHB, EPB41L5, RACGAP1, MRPS23, TOP2A, H2AFJ, NOL3, MIEN1, CENPW, LY6E, ALYREF, MMP1, and NDUFB10, the pT and the pN;
(e) stratifying the subject based upon the calculated risk score; and
(f) administering at least one treatment to the subject based on the risk score, wherein the treatment comprises surgery, radiation, anthracycline agents, alkylating agents, nucleoside analogs, platinum agents, vinca agents, anti-estrogen drugs, aromatase inhibitors, ovarian suppression agents, endocrine/hormonal agents, bisphophonate therapy agents, cyclophosphamide, fluorouracil (or 5-fluorouracil or 5-FU), methotrexate, thiotepa, carboplatin, cisplatin, gemcitabine, anthracycline, taxanes, paclitaxel, protein-bound paclitaxel, doxorubicin, docetaxel, vinorelbine, tamoxifen, raloxifene, toremifene, fulvestrant, irinotecan, ixabepilone, temozolmide, topotecan, vincristine, vinblastine, eribulin, mutamycin, capecitabine, capecitabine, anastrozole, exemestane, letrozole, leuprolide, abarelix, buserlin, goserelin, megestrol acetate, risedronate, pamidronate, ibandronate, alendronate, denosumab, zoledronate, trastuzumab, tykerb or bevacizumab, or combinations thereof,
wherein the risk score is calculated according to the following formula:
Risk
⁢
Score
=
(
0
.
0
⁢
3
⁢
6
⁢
0
⁢
4
)
×
(
∑
i
⁢
β
i
×
Cq
normalized
)
+
pT
+
pN
5
.
6
⁢
4
⁢
0
⁢
3
⁢
2
wherein
i is the summation index for the genes for which the mRNA expression level was measured;
β is the ridge penalized Cox model coefficient for each gene as set forth below:
H2AFZ-0.03833591325196550, CDK1-0.06132455806571770, EXOSC4-0.02105976326055420, PHLDA2-0.06295739658169650, APOBEC3B 0.02341881674020150, EIF4EBP1-0.13911217901125500, SEN 0.05788269046891110, PHB-0.03538557745953510, EPB41L5-0.04675539403890050, RACGAP1-0.05097505893853430, MRPS23-0.14201022110072700, TOP2A-0.11290078348786600, H2AFJ-0.04975471358452700, NOL3-0.04193802459521500, MIEN1 0.01133668644106850, CENPW-0.03717918353187610, LY6E-0.02829256296234230, ALYREF-0.09541915699494330, MMP1-0.00911370427072023, NDUFB10 0.00626166874136819;
and Cqnormalized is the normalized average quantification cycle (Cq) for each of the genes for which the mRNA expression level was measured;
wherein
pT is 0.45542 when the tumor size of the at least one ER+/HER2− breast cancer tumor is determined to be pT1c; or
pT is 0.96572 when the tumor size of the ER+/HER2− breast cancer tumor is determined to be any of pT2, pT3 and pT4; and
wherein
pN is 0.46293 when the nodal status of the at least one ER+/HER2− breast cancer tumor is determined to be pN1 or
pN is 1.24922 when the nodal status of the at least one ER+/HER2− breast cancer tumor is determined to be pN2.

### Claim 15 (depends on 14)

The method of claim 14, wherein stratifying the subject based upon the calculated risk score comprises (a) stratifying the subject as having a low risk for ER+/HER2− breast cancer recurrence when the risk score is less than a first predetermined cutoff value; (b) stratifying the subject as having a high risk for ER+/HER2− breast cancer recurrence when the risk score is greater than a second predetermined cutoff value and/or (c) stratifying the subject as having an intermediate risk for ER+/HER2− breast cancer recurrence when the risk score is greater than or equal to a first predetermined cutoff value and less than or equal to a second predetermined cutoff value.

### Claim 16 (depends on 14)

The method of claim 14, wherein Cqnormalized is normalized to the expression of at least two reference genes.

### Claim 17 (depends on 16)

The method of claim 16, wherein the at least two reference genes are
(a) housekeeping genes or
(b) selected from the group consisting of GAPDH, GUSB, HPRTI, and TBP.

### Claim 18 (depends on 14)

The method of claim 14, wherein Cqnormalized is calculated according to the following formula:
Cq
normalized
=
AVG
⁢
Cq
-
SF
wherein SF is the difference between the AVG Cq value of the at least two reference genes for each subject and a constant reference value K.

### Claim 19 (depends on 18)

The method of claim 18, wherein (a) K is the mean of the AVG Cq of the at least two reference genes calculated across a plurality of training samples or (b) wherein K=25.012586069.

### Claim 20 (depends on 14)

The method of claim 14, wherein the mRNA expression levels are determined using one or more techniques selected from the group consisting of analysis of single strand conformation polymorphism, capillary electrophoresis, denaturing high performance liquid chromatography, digital molecular barcoding technology, direct sequencing, DNA mismatch-binding protein assays, dynamic allele-specific hybridization, Fluorescent in situ hybridization (FISH), high-density oligonucleotide SNP arrays, high-resolution melting analysis, microarray, next generation sequencing (NGS), Northern blot analysis, nuclease protection analysis, oligonucleotide ligase assays, polymerase chain reaction (PCR), primer extension assays, Quantigene analysis, quantitative nuclease-protection assay (qNPA), reporter gene detection, restriction fragment length polymorphism (RFLP) assays, reverse transcription and real-time quantitative polymerase chain reaction (RT-qPCR), reverse transcription-polymerase chain reaction (RT-PCR), RNA sequencing (RNA-seq), Serial analysis of gene expression (SAGE), Single Molecule Real Time (SMRT) DNA sequencing technology, SNPLex, Southern blot analysis, Sybr Green chemistry, TaqMan-based assays, temperature gradient gel electrophoresis (TGGE), and Tiling array.