Office Action 2: Notice of Allowance (2025-02-12)

CLAIMS:
AMENDMENTS TO THE CLAIMS

This listing of claims will replace all prior versions, and listings, of claims in this application.

1\. (Cancelled)
2\. (Currently Amended) A method, comprising:
  treating DNA from a sample with a reagent that modifies DNA in a methylation-specific manner;
amplifying the treated DNA using a set of primers specific for each o four or more genes, wherein the four or more genes comprises FAM59B, SPHK2, ODC1, and at least one of OSR2 A, OSR2 B, or OSR2 C or any combinations thereof; anddetermining \[\[the\]\]a methylation level ofeach of the four or more genes usingpolymerase chain reaction, nucleic acid sequencing, mass spectrometry, a methylation-specific nuclease, mass-based separation, and/or target capture;SVG {EMBED HERE} 0.1130.09
3\. (Cancelled)
4\. (Currently Amended) The method of claim \[\[3\]\]2, wherein the reagent comprises one or more of a methylation-sensitive restriction enzyme, a methylation-dependent restriction enzyme, and/or a bisulfite reagent.
5\. (Previously Presented) The method of claim 4, wherein the DNA is treated with a bisulfite reagent to produce bisulfite-treated DNA.
6\. (Currently Amended) The method of claim 2, wherein determining the methylation level of each of the four or more genes comprises multiplex amplification.

7\. (Currently Amended) The method of claim 2, wherein determining the methylation level of each of the four or more genes comprises using methylation-specific PCR, quantitative methylation-specific PCR, methylation-specific DNA restriction enzyme analysis, quantitative bisulfite pyrosequencing, a flap endonuclease assay,aPCR-flap assay, and/or bisulfite genomic sequencing PCR.

8\. (Cancelled)

9\. (Currently Amended) The method of claim 2, wherein:the set of primers specific for FAM59B fcompriseSEQ ID \[\[NOS\]\]NOs: 299 and 300 or SEQ ID \[\[NOS\]\]NOs: 427 and 428\[\[,\]\]i the set of primers specific for ODCIfcompriseSEQ ID \[\[NOS\]\]NOs: 181 and 182, SEQ ID \[\[NOS\]\]NOs: 183 and 184, SEQ ID \[\[NOS\]\]NOs: 185 and 186, SEQ ID \[\[NOS\]\]NOs: 341 and 342, or SEQ ID \[\[NOS\]\]NOs: 443 and 444\[\[,\]\];
the set of primers specific for OSR2 A compriseSEQ ID \[\[NOS\]\]NOs: 187 and 188, SEQ ID \[\[NOS\]\]NOs: 343 and 344, or SEQ ID \[\[NOS\]\]NOs: 445 and 446\[\[,\]\]; and
the set of primers specific for SPHK2 compriseSEQ ID \[\[NOS\]\]NOs: 225 and 226, SEQ ID \[\[NOS\]\]NOs: 355 and 356, SEQ ID \[\[NOS\]\]NOs: 337 and 338, or SEQ ID \[\[NOS\]\]NOs: 433 and 434.

10\. (Previously Presented) The method of claim 2, wherein the sample comprises tissue.

11\. (Previously Presented) The method of claim 10, wherein the tissue is breast tissue.

12\. (Currently Amended) The method of claim 2, wherein the sample is ablood sample, a serum sample, or a plasma sample.

OFFICE ACTION:
Notice of Pre-AIA  or AIA  Status
The present application, filed on or after March 16, 2013, is being examined under the first inventor to file provisions of the AIA .
Response to Request for Information
The response filed December 19, 2024 provides the following response to the request for information.
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The examiner has reviewed the references deemed relevant by Applicant.
Reasons for Allowance
The following is an examiner’s statement of reasons for allowance:
The claims are directed to specifically amplifying DNA treat to modify the DNA in a methylation -specific manner using primers specific for four or more genes, namely SPHK2, ODC1, FAM59B and an OSR2, as recited in the claims to determine the methylation level using PCR, sequencing, mass spectrometry, methylation-specific nuclease, mass-based separation and/or target capture.
The claims require using primers that are specifically directed to each of the recited four genes and thus do not read on simple array-based method using a commercially available array without first specific amplifying for the four genes.
Any comments considered necessary by applicant must be submitted no later than the payment of the issue fee and, to avoid processing delays, should preferably accompany the issue fee.  Such submissions should be clearly labeled “Comments on Statement of Reasons for Allowance.”
Conclusion

APPLICANT REBUTTAL: REMARKS
1. Status of the Claims
Claims 2-12 were pending in this application. Claim I was previously cancelled. As provided herein, claims 2, 4, 6, 7, 9, and 12 have been amended; and claims 3 and 8 have been cancelled. Therefore, claims 2, 4-7, and 9-12 are currently under consideration. No new matter has been added.
Applicant requests entry of the remarks made herein into the file history of the application and reserves the right to file a divisional or continuation application to any unclaimed subject matter.
2. Patentability Remarks
37 C. F.R § 1.105 - Request for Information
On pp. 2-3 of the Office Action, the Examiner asserts: "In total, the IDS filed prior to the writing of this first action on the merits contains
,
references on 28
pag
es of IDS.
The extraordinary number of references places a serious burden on the office. The examiner finds that not each one of the greater than 310 references listed on the IDS are relevant to the instantly claimed invention which requires only measuring the methylation level of CpG sites in FAM59B, OSR2, SPHK2, ODCl. For example, NPL reference 57 of the IDS, "Detection of occult upper gastrointestinal tract bleeding:performance differences in fecal occult blood tests', NPL reference 89 of the IDS, 'Revision of consensus sequence of human Alu repeats: a review (1987)', NPL reference 100 of the IDS, 'Stool DNA screening for colorectal cancer: opportunities to improve value with the next generation tests (2011)'. Therefore, the burden is shifted to the applicants and they are specifically required to point out the references cited which are most relevant to instant claims. While art has been applied in this first action on the merits, the IDS filed March 18, 2021 and December 1, 2023 has an enormous number of references, and so this requirement for information is set forth" (emphasis in original).
Additionally, on p. 3 of the Office Action, the Examiner asserts that the following information is required from Applicant: "A) Which references disclose methods of detecting methylation of FAM59B, OSH2, SPHK2 and/or ODCI? B) Which references teach that methylation of FAM59B, OSR2, SPHK2 and/or ODC1 is found in breast cancer or breast disorders?"
Applicant submits that all currently cited IDS references for the present application were cited to comply with Applicant's duty of candor and good faith under 37 C.F.R. § 1.56.
Applicant does not believe that the cited references place an undue burden on the Examiner or the UPSTO. Moreover, a request for information under 37 C.F.R. § 1.105 does not impose an obligation upon the Applicant to conduct a search on behalf of the Examiner.
Nevertheless, solely to comply with the Examiner's request, Applicant compiled a searchable database containing the references cited in the Nonpatent Literature Documents and Foreign Patent Documents sections of the IDSs submitted for the present application (including the current IDS submitted herewith). Using widely accessible document management software (i.e., iManage Work 10), Applicant conducted the following search of this database using Boolean operators to comply with part (A) of the Examiner's request: ["FAM59B" OR "SPHK2" OR "ODC1" OR "OSR2"]. Performance of this search yielded the following references:
W02021041726A1,
W02020112869A1,
W02019108626A1,
W02017192221A1,
W02022165247A1,
W02021226074A2,
W02019068082A1,
W02017040627A1, W020 14159652, W020 1203 1008A2, and W02008079269.
While Applicant has supplied the above search results in good faith solely to comply with the Examiner's request in this specific instance, Applicant respectfully submits that such Boolean operator searches for a defined set of markers within digitally searchable references are well within the advanced searching capabilities of the USPTO's Patent Search System and do not justify shifting the Examiner's obligations to Applicant.
Regarding part (B) of the Examiner's request, Applicant submits that "breast cancer" or "breast disorders" are terms that are not present in the currently pending claims. Therefore, Applicant respectfully submits that the Examiner's request for the information in part (B) of the request is beyond the scope of 37 C.F.R. § 1.105. Furthennore, if deemed necessary for assessing the claimed subject matter, Applicant submits that appropriate searches can readily be performed by the Examiner using USPTO resources.
Taken together, Applicant respectfully submits that the Examiner's request under 37 C.F.R. § 1.105 has been satisfied.
35 U.S.C. § 112 - New Matter
On pp. 3-4 of the Office Action, claims 2-12 were rejected for allegedly containing new matter for recitation of the term "OSR2." Applicant respectfully disagrees; however, without acquiescing to the rejections, or the underlying rationale presented in the Office Action for the rejections, and solely to advance prosecution, independent claim 2 has been amended to recite, inter alia, "amplifying the treated DNA using a set of primers specific for each of four or more genes, wherein the four or more genes comprises FAM59B, SPHK2, ODC1, and at least one
of OSR2_A, OSR2_B, and/or OSR2_C." Accordingly, Applicant respectfully requests that these rejections be withdrawn.
35 U.S.C. § 112(b) - Indefiniteness
On pp. 4-6 of the Office Action, the Examiner rejects claims 2-12 under 35 U.S.C. § 112(b) for allegedly being indefinite for recitation of the term "OSR2." Applicant respectfully disagrees; however, independent claim 2 has been amended as described above. Accordingly, Applicant respectfully requests that this rejection be withdrawn.
Additionally, the Examiner rejected claim 8 under 35 U.S.C. § 112(b) for allegedly being indefinite for recitation of chromosomal coordinates. Applicant respectfully disagrees; however, without acquiescing to the rejections, or the underlying rationale presented in the Office Action for the rejections, and solely to advance prosecution, claim 8 has been cancelled. Therefore, this rejection has been rendered moot.
35 U.S.C. § 112(4) - Claim Dependency
On p. 6 of the Office Action, the Examiner rejects claim 3 under 35 U.S.C. § 112(d) for allegedly being of improper dependent form. Applicant respectfully disagrees; however, without acquiescing to the rejection, or the underlying rationale presented in the Office Action for the rejection, and solely to advance prosecution, claim 3 has been cancelled. Therefore, Applicant respectfully requests that these rejections be withdrawn. Therefore, this rejection has been rendered moot.
CONCLUSION


CITED REFERENCE SUMMARIES: Ref US20070059753: Claims:
A method of diagnosing the presence or predicting the course of a proliferative disorder comprising determining the methylation status of a Marker in a biological sample with the use of a reagent selected from the group consisting of: Seq. ID NO. 1-35 and 44-53.
The method according to claim 1 further comprising measuring the presence of a reference Marker.
The method according to claim 2 wherein the reference Marker is selected from the group consisting of B Actin.
The method of claim wherein a combination of Markers are used.
The method of claim 4 wherein the combination of Markers includes a Marker for GSTP1 and a Marker for APC, TIMP, RASSF1A, RARB2, PTGS2, or 14-3-3.
The method according to claim 4 wherein reagents for the Markers include a member of the group consisting of Seq. ID. No. 1-35 and 44-53.
The method of claim 1 wherein the sample is prostate tissue.
The method of claim 1 wherein the sample is urine, serum, plasma, or circulating cells.
A composition comprising a member of the group consisting of Seq. ID No. 1-43.
A composition comprising a mixture of more than one composition of claim 10 .
A kit for conducting an assay according to claim 1 , comprising: nucleic acid amplification and detection reagents.
The kit of claim 11 wherein the amplification and detection reagents are selected from the group consisting of Seq. ID No. 1-43.
The kit of claim 12 wherein the amplification and detection reagents are selected from Seq. ID No. 1-25 together with reagents selected from the group consisting of Seq. ID No. 36-43.
The kit of claim 11 further comprising instructions.
The kit of claim 11 wherein the reagents detect the hypermethylation of a gene selected from the group consisting of GSTP1 and APC, TIMP, RASSF1a, RARB2, PTGS2, or 14-3-3.
The kit of claim 11 further comprising reagents for amplifying and detecting the presence of a constitutively expressed gene.
The kit of claim 11 further comprising reagents for amplifying and detecting the presence of constitutively expressed genes.
The method of claim 1 further comprising establishing a methylation ratio and determining whether the methylation ratio exceeds a cutoff value.
The method of claim 1 for use in therapy monitoring.
The method of claim 1 for use in screening.
The method of claim 1 for use in resolving ambiguous or indeterminate results of cancer assays obtained using methods different from claim 1.
Ref US20080044824: Claims:
A method for characterizing thyroid tissue, comprising: a) providing a thyroid tissue sample from a subject; and b) detecting the level of expression of kallikrein 10 is said sample.
a) providing a thyroid tissue sample from a subject; and
b) detecting the level of expression of kallikrein 10 is said sample.
The method of claim 1 , wherein said detecting the level of expression of kallikrein 10 in said sample comprises detecting the level of expression of kallikrein 10 mRNA.
The method of claim 2 , wherein said detecting the level of expression of kallikrein 10 mRNA comprises exposing said mRNA to a nucleic acid probe complementary to said mRNA.
The method of claim 2 , wherein said detecting the level of expression of kallikrein 10 mRNA comprises performing a Q-RT-PCR assay.
The method of claim 1 , wherein said detecting said detecting the level of expression of kallikrein 10 comprises detecting the level of expression of a kallikrein 10 polypeptide.
The method of claim 5 , wherein said detecting the level of expression of a kallikrein 10 polypeptide comprises exposing said polypeptide to an antibody specific to said polypeptide and detecting the binding of said antibody to said polypeptide.
The method of claim 6 , wherein said detecting comprises immunohistochemistry.
The method of claim 1 , wherein an increased level of expression of said kallikrein 10 gene relative to the level of expression of said kallikrein 10 gene in a non-cancerous control is indicative of thyroid cancer in said sample.
The method of claim 1 , wherein said subject comprises a human subject.
The method of claim 1 , wherein said sample comprises tumor tissue.
The method of claim 1 , wherein said characterizing said thyroid tissue comprises identifying a type of thyroid cancer in said thyroid tissue.
The method of claim 11 , wherein said type of thyroid cancer is selected from the group consisting of papillary, follicular, medullary, and anaplastic.
The method of claim 1 , further comprising the step of d) providing a diagnosis to said subject.
The method of claim 13 , wherein said diagnosis comprises a diagnosis of thyroid cancer.
A method for characterizing thyroid tissue, comprising: a) providing a thyroid tissue sample from a subject; and b) detecting the level of expression of claudin 1 in said sample.
b) detecting the level of expression of claudin 1 in said sample.
The method of claim 15 , wherein an increased level of expression of said claudin 1 gene relative to the level of expression of said claudin 1 gene in a non-cancerous control is indicative of papillary carcinoma in said sample.
The method of claim 15 , wherein said detecting the level of expression of said claudin 1 gene in said sample comprises detecting the level of expression of claudin 1 mRNA.
A method for characterizing thyroid tissue, comprising: a) providing a thyroid tissue sample from a subject; and b) detecting the level of expression of a gene selected from the group consisting of PPARG, ATP10B, C6orf29, CHIA, CNR1, AQP7, ANGPTL4, ENO3, TFPI2, MFAP3L, IGFBP2, FBP1, SLC19A1, FBN2, LEPREL1, RAB15, TNFRSF21, CHRNA7, RASSF4, MYOZ1, CCL14, FGFBP1 REPS2, ACAA1, SCNN1A, ARID5B, MYCL1, GFAP, GPR109B, SSX2, XK, DNASE1, SCNN1B, SLC7A8, S100A5, APBB2, DHCR24, SLC39A8, FAM59A, ANKS1, CMKOR1, GENX-3414, FAT2, PLS1, HIC2, GRK5, RASL11B, FDFT1, DECR2, SSX1, ITPR1, SSX3, PMP22, C2orf31, RSU1, PTTG1, ENO2, SALL1, FLJ11196, MAN1C1, PLEKHB1, PTTG3, CUEDC1, MXI1, CHST5, C7orf32, CHPT1, and EPHB2 in said sample.
b) detecting the level of expression of a gene selected from the group consisting of PPARG, ATP10B, C6orf29, CHIA, CNR1, AQP7, ANGPTL4, ENO3, TFPI2, MFAP3L, IGFBP2, FBP1, SLC19A1, FBN2, LEPREL1, RAB15, TNFRSF21, CHRNA7, RASSF4, MYOZ1, CCL14, FGFBP1 REPS2, ACAA1, SCNN1A, ARID5B, MYCL1, GFAP, GPR109B, SSX2, XK, DNASE1, SCNN1B, SLC7A8, S100A5, APBB2, DHCR24, SLC39A8, FAM59A, ANKS1, CMKOR1, GENX-3414, FAT2, PLS1, HIC2, GRK5, RASL11B, FDFT1, DECR2, SSX1, ITPR1, SSX3, PMP22, C2orf31, RSU1, PTTG1, ENO2, SALL1, FLJ11196, MAN1C1, PLEKHB1, PTTG3, CUEDC1, MXI1, CHST5, C7orf32, CHPT1, and EPHB2 in said sample.
The method of claim 18 , wherein an altered level of expression of said gene in said sample relative to the level of expression of said gene in a non-cancerous control is indicative of follicular thyroid carcinoma in said sample.
The method of claim 18 , wherein said detecting the level of expression of said gene in said sample comprises detecting the level of expression of mRNA from said gene.
Ref US20120172238: Claims:
Method for assisting in diagnosing breast cancer and/or monitoring breast cancer progression, comprising: (a) determining a methylation state at one or more genomic loci of the DNA comprised in a given sample to be analyzed; (b) identifying one or more genomic loci exhibiting differences in its/their DNA methylation state; (c) performing a statistical survival analysis for each of the one or more differentially methylated genomic loci obtained in step (b); (d) determining a statistical significance of the data obtained in step (c); and (e) selecting one or more genomic loci displaying statistically significant differences in its/their DNA methylation state based on the data obtained in step (d), wherein the one or more genomic loci selected have prognostic value for assisting in diagnosing breast cancer and/or monitoring breast cancer progression.
(a) determining a methylation state at one or more genomic loci of the DNA comprised in a given sample to be analyzed;
(b) identifying one or more genomic loci exhibiting differences in its/their DNA methylation state;
(c) performing a statistical survival analysis for each of the one or more differentially methylated genomic loci obtained in step (b);
(d) determining a statistical significance of the data obtained in step (c); and
(e) selecting one or more genomic loci displaying statistically significant differences in its/their DNA methylation state based on the data obtained in step (d), wherein the one or more genomic loci selected have prognostic value for assisting in diagnosing breast cancer and/or monitoring breast cancer progression.
The method of claim 1 , wherein the breast cancer is estrogen receptor positive breast cancer.
The method of claim 1 for assisting in diagnosing breast cancer and/or monitoring breast cancer progression in a patient, further comprising: providing a genomic DNA sample from the patient to be analyzed, wherein the method is performed in vitro.
providing a genomic DNA sample from the patient to be analyzed,
wherein the method is performed in vitro.
The method of claim 1 , further comprising: classifying the one or more genomic loci according to its/their methylation state as unmethylated, partially methylated, and methylated prior to performing step (c).
classifying the one or more genomic loci according to its/their methylation state as unmethylated, partially methylated, and methylated prior to performing step (c).
The method of claim 1 , wherein the statistical survival analysis performed in step (c) comprises generating Kaplan-Meier survival estimates for the respective methylation states of each of the one or more genomic loci and calculating the differences between the Kaplan-Meier survival estimates generated for each of the loci.
The method of claim 1 , wherein determining the statistical significance of the data obtained in the survival analysis comprises applying the log-rank or Mantel-Haenszel test.
The method of claim 6 , wherein determining the statistical significance further comprises a permutation testing method.
The method of claim 1 , further comprising: determining whether the prognostic value of the one or more genetic loci selected is independent of other pathological parameters than the methylation state.
determining whether the prognostic value of the one or more genetic loci selected is independent of other pathological parameters than the methylation state.
The method of claim 1 , wherein the method is performed using a computing device.
Panel of genetic markers for assisting in diagnosing breast cancer and/or monitoring breast cancer progression in a patient, wherein the panel comprises any one or more, combination thereof, or all, of the genetic markers listed in Table 1.
Panel of genetic markers for assisting in diagnosing estrogen receptor positive breast cancer and/or monitoring estrogen receptor positive breast cancer progression in a patient, wherein the panel comprises any one or more, preferably combination thereof, or all, of the genetic markers listed in Table 2.
The panel of genetic markers of claim 10 or 11 , wherein the panel is determined by the method as defined in any of claims 1 to 9 .
The panel of claim 10 , wherein monitoring breast cancer progression comprises stratification of breast cancer patients into good or poor prognosis groups.
The panel of claim 10 , wherein monitoring breast cancer progression comprises predicting relapse free survival at five years from diagnosis.
Ref US20190161805: Claims:
A method, comprising: measuring a methylation level for one or more genes in a biological sample of a human individual through treating genomic DNA in the biological sample with a reagent that modifies DNA in a methylation-specific manner; amplifying the treated genomic DNA using a set of primers for the selected one or more genes; and determining the methylation level of the one or more genes by polymerase chain reaction, nucleic acid sequencing, mass spectrometry, methylation-specific nuclease, mass-based separation, and target capture; wherein the one or more genes comprises a chromosomal region having an annotation that is selected from one of the following groups: (i) ATP6V1B1, LMX1B_A, BANK1, OTX1, MAX.chr11.14926602-14927148, UBTF, PRKCB, TRH_A, MPZ, DNM3_A, TRIM67, MAX.chr12.4273906-4274012, CALN1_A, ITPRIPL1, MAX.chr12.4273906-4274012, GYPC_B, MAX.chr5.42994866-42994936, OSR2_A, SCRT2_B, MAX.chr5.145725410-145725459, MAX.chr11.68622869-68622968, MAX.chr8.124173030-124173395, MAX.chr20.1784209-1784461, LOC100132891, BHLHE23_D, MAX.chr19.46379903-46380197, CHST2_B, MAX.chr5.77268672-77268725, C17orf64, EMX1_A, DSCR6, ITPRIPL1, IGF2BP3_B, DLX4, and ABLIM1; (ii) ABLIM1_B, AJAP1_C, ALOX5_B, ASCL2_B, BANK1_B, BHLHE23_E, C10orf125_B, C17orf64_B, CALN1_1520, CALN1_B, CD1D_1058, CDH4_7890, CHST2_8128, CHST2_8384, CHST2_9316, CHST2_9470, CLIC6_B, CXCL12_B, DLX4_B, DNM3_D, EMX1_A, ESPN_B, FAM59B_7764, FOXP4_B, GP5, HOXA1_C, IGF2BP3_C, IPTRIPL1_1138, IPTRIPL1_1200, KCNK9_B, KCNK17_C, LAYN_B, LIME1_B, LMX1B_D, LOC100132891_B, MAST1_B, MAX.chr12.427.br, MAX.chr20.4422, MPZ_5742, MPZ_5554, MSX2P1_B, ODC1_B, OSR2_A, OTX1_B, PLXNC1_B, PRKCB_7570, SCRT2_C, SLC30A10, SPHK2_B, ST8SIA4_B, STX16_C, TRH_A, and TRIM67_B; and (iii) CD1D, ITPRIPL1, FAM59B, C10orf125, TRIM67, SPHK2, CALN1_B, CHST2_B, MPZ, CXCL12_B, ODC1_B, OSR2_A, TRH_A, and C17orf64_B.
measuring a methylation level for one or more genes in a biological sample of a human individual through treating genomic DNA in the biological sample with a reagent that modifies DNA in a methylation-specific manner; amplifying the treated genomic DNA using a set of primers for the selected one or more genes; and determining the methylation level of the one or more genes by polymerase chain reaction, nucleic acid sequencing, mass spectrometry, methylation-specific nuclease, mass-based separation, and target capture; wherein the one or more genes comprises a chromosomal region having an annotation that is selected from one of the following groups: (i) ATP6V1B1, LMX1B_A, BANK1, OTX1, MAX.chr11.14926602-14927148, UBTF, PRKCB, TRH_A, MPZ, DNM3_A, TRIM67, MAX.chr12.4273906-4274012, CALN1_A, ITPRIPL1, MAX.chr12.4273906-4274012, GYPC_B, MAX.chr5.42994866-42994936, OSR2_A, SCRT2_B, MAX.chr5.145725410-145725459, MAX.chr11.68622869-68622968, MAX.chr8.124173030-124173395, MAX.chr20.1784209-1784461, LOC100132891, BHLHE23_D, MAX.chr19.46379903-46380197, CHST2_B, MAX.chr5.77268672-77268725, C17orf64, EMX1_A, DSCR6, ITPRIPL1, IGF2BP3_B, DLX4, and ABLIM1; (ii) ABLIM1_B, AJAP1_C, ALOX5_B, ASCL2_B, BANK1_B, BHLHE23_E, C10orf125_B, C17orf64_B, CALN1_1520, CALN1_B, CD1D_1058, CDH4_7890, CHST2_8128, CHST2_8384, CHST2_9316, CHST2_9470, CLIC6_B, CXCL12_B, DLX4_B, DNM3_D, EMX1_A, ESPN_B, FAM59B_7764, FOXP4_B, GP5, HOXA1_C, IGF2BP3_C, IPTRIPL1_1138, IPTRIPL1_1200, KCNK9_B, KCNK17_C, LAYN_B, LIME1_B, LMX1B_D, LOC100132891_B, MAST1_B, MAX.chr12.427.br, MAX.chr20.4422, MPZ_5742, MPZ_5554, MSX2P1_B, ODC1_B, OSR2_A, OTX1_B, PLXNC1_B, PRKCB_7570, SCRT2_C, SLC30A10, SPHK2_B, ST8SIA4_B, STX16_C, TRH_A, and TRIM67_B; and (iii) CD1D, ITPRIPL1, FAM59B, C10orf125, TRIM67, SPHK2, CALN1_B, CHST2_B, MPZ, CXCL12_B, ODC1_B, OSR2_A, TRH_A, and C17orf64_B.
treating genomic DNA in the biological sample with a reagent that modifies DNA in a methylation-specific manner;
amplifying the treated genomic DNA using a set of primers for the selected one or more genes; and
determining the methylation level of the one or more genes by polymerase chain reaction, nucleic acid sequencing, mass spectrometry, methylation-specific nuclease, mass-based separation, and target capture; wherein the one or more genes comprises a chromosomal region having an annotation that is selected from one of the following groups:
wherein the one or more genes comprises a chromosomal region having an annotation that is selected from one of the following groups:
(i) ATP6V1B1, LMX1B_A, BANK1, OTX1, MAX.chr11.14926602-14927148, UBTF, PRKCB, TRH_A, MPZ, DNM3_A, TRIM67, MAX.chr12.4273906-4274012, CALN1_A, ITPRIPL1, MAX.chr12.4273906-4274012, GYPC_B, MAX.chr5.42994866-42994936, OSR2_A, SCRT2_B, MAX.chr5.145725410-145725459, MAX.chr11.68622869-68622968, MAX.chr8.124173030-124173395, MAX.chr20.1784209-1784461, LOC100132891, BHLHE23_D, MAX.chr19.46379903-46380197, CHST2_B, MAX.chr5.77268672-77268725, C17orf64, EMX1_A, DSCR6, ITPRIPL1, IGF2BP3_B, DLX4, and ABLIM1;
(ii) ABLIM1_B, AJAP1_C, ALOX5_B, ASCL2_B, BANK1_B, BHLHE23_E, C10orf125_B, C17orf64_B, CALN1_1520, CALN1_B, CD1D_1058, CDH4_7890, CHST2_8128, CHST2_8384, CHST2_9316, CHST2_9470, CLIC6_B, CXCL12_B, DLX4_B, DNM3_D, EMX1_A, ESPN_B, FAM59B_7764, FOXP4_B, GP5, HOXA1_C, IGF2BP3_C, IPTRIPL1_1138, IPTRIPL1_1200, KCNK9_B, KCNK17_C, LAYN_B, LIME1_B, LMX1B_D, LOC100132891_B, MAST1_B, MAX.chr12.427.br, MAX.chr20.4422, MPZ_5742, MPZ_5554, MSX2P1_B, ODC1_B, OSR2_A, OTX1_B, PLXNC1_B, PRKCB_7570, SCRT2_C, SLC30A10, SPHK2_B, ST8SIA4_B, STX16_C, TRH_A, and TRIM67_B; and
(iii) CD1D, ITPRIPL1, FAM59B, C10orf125, TRIM67, SPHK2, CALN1_B, CHST2_B, MPZ, CXCL12_B, ODC1_B, OSR2_A, TRH_A, and C17orf64_B.
The method of claim 1 , wherein the DNA is treated with a reagent that modifies DNA in a methylation-specific manner.
The method of claim 2 , wherein the reagent comprises one or more of a methylation-sensitive restriction enzyme, a methylation-dependent restriction enzyme, and a bisulfite reagent.
The method of claim 3 , wherein the DNA is treated with a bisulfite reagent to produce bisulfite-treated DNA.
The method of claim 1 , wherein the measuring comprises multiplex amplification.
The method of claim 1 , wherein measuring the amount of at least one methylated marker gene comprises using one or more methods selected from the group consisting of methylation-specific PCR, quantitative methylation-specific PCR, methylation-specific DNA restriction enzyme analysis, quantitative bisulfite pyrosequencing, flap endonuclease assay, PCR-flap assay, and bisulfite genomic sequencing PCR.
The method of claim 1 , wherein the sample comprises one or more of a plasma sample, a blood sample, or a tissue sample (e.g., breast tissue).
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 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.chr11.14926602-14927148 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 selected from the group consisting of SEQ ID NOS: 175 and 176 and SEQ ID NOS: 439 and 440, 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 MAX.chr12.4273906-4274012 a set of primers selected from the group consisting of SEQ ID NOS: 133 and 134 and SEQ ID NOS: 135 and 136, for CALN1_A a set of primers consisting of SEQ ID NOS: 29 and 30, for ITPRIPL1 a set of primers selected from the group consisting of from SEQ ID NOS: 97 and 98, SEQ ID NOS: 99 and 100 and SEQ ID NOS: 425 and 426, 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.chr11.68622869-68622968 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 o
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