DETAILED 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 .
Election/Restrictions
Applicant’s election without traverse Species Group I, Species A, claims 9-10 and Species Group II, Species B, claim 37 in the reply filed on 02/18/2026 is acknowledged.
Claim Status
Claims 1-13 and 21-37 are pending.
Claims 1-10, 12, 21-35, and 37 are examined on the merit.
Claims 11,13 and 36 are withdrawn from further consideration.
Claims 14-20 are canceled.
Priority
The instant application claims the benefit of priority as a continuation of U.S. Application No. 16/903,231 filed 12/28/2020, and 16/389/753 filed on 04/19/2019 and 14/495,791 filed 24 September 2014. The ‘791 application claims the benefit of priority as a continuation of International Application No. PCT/AU2013/001088 filed 20 September 2013. The claim to the benefit of priority as a continuation of U.S. Application No. 14/495,791 filed 24 September 2014 as set forth on the Application Data Sheet and International Application No. PCT/AU2013/001088 filed 20 September 2013 is acknowledged.
The ‘088 applications claim the benefit of priority to U.S. Provisional Application No. 61/830,571 field 3 June 2013 and as a continuation in part of U.S. Application No. 13/842,209 filed 15 March 2013. The ‘209 application further claims the benefit of priority to U.S. Provisional Application No. 61/703,512 filed 20 September 2012.
Claim 1, and those claims dependent therefrom, recite calculating a first methylation level based on respective numbers of cell-free DNA molecules that are hypermethylated at the plurality of sites. This limitation is not supported by the ‘512 application. Therefore, claims 1-10, 12, 21-35, and 37 are not granted the claim to the benefit of priority to the ‘512 application.
Claim 5 recites treating the cell-free DNA molecules with sodium bisulfite as part of Tet- assisted bisulfite conversion or oxidative bisulfite sequencing. This limitation is not support by the ‘512, ‘209 or ‘571 applications. Therefore, claim 5 is not granted the claim to the benefit of priority to the ‘512, ‘209 and ‘571 applications.
Claims 21-22 recite limitations for analyzing the hypermethylation of CpG islands. These limitations are not supported by the disclosure of the ‘512 or ‘209 Applications. Therefore, claims 21-22 and 25 are not granted the claim to the benefit of priority to the ‘512 and ‘209 Applications.
In this action, all claims are examined as though they had the above-mentioned effective filing dates. In future actions, the effective filing date of one or more claims may change, due to amendments to the claims, or further analysis of the disclosure(s) of the priority application(s).
Information Disclosure Statement
The information disclosure statements (IDS) submitted on 09/04/2025 and 11/18/2025 are in compliance with the provisions of 37 CFR 1.97. Accordingly, the list of cited references was considered in full by the examiner. A signed copy of the corresponding 1449 form has been included with this Office action.
Drawings
The drawings filed 07/17/2025 are accepted.
Specification
The amendments to the specification filed 07/17/2025 have been accepted.
Claim Rejections - 35 USC § 112
The following is a quotation of the first paragraph of 35 U.S.C. 112(a):
(a) IN GENERAL. —The specification shall contain a written description of the invention, and of the manner and process of making and using it, in such full, clear, concise, and exact terms as to enable any person skilled in the art to which it pertains, or with which it is most nearly connected, to make and use the same, and shall set forth the best mode contemplated by the inventor or joint inventor of carrying out the invention.
Claim 29 is rejected under 35 U.S.C. 112(a) or 35 U.S.C. 112 (pre-AIA ), first paragraph, as failing to comply with the enablement requirement. The claim(s) contains subject matter which was not described in the specification in such a way as to enable one skilled in the art to which it pertains, or with which it is most nearly connected, to make and/or use the invention.
The claim recitation of “a non-transitory computer readable medium comprising a plurality of instructions that, when executed, control a computer system to perform a methylation-aware assay comprising enrichment of cell-free DNA molecules originating from specific genomic regions, wherein the specific genomic regions comprise genomic regions with a methylation differential between cancer and non-cancer for a tissue, wherein the enrichment comprises:(i) contacting the plurality of cell-free DNA molecules with hybridization probes to enrich for cell-free DNA molecules originating from the specific genomic regions, and determining sequences of the enriched cell-free DNA molecules; or (ii) performing methylation-specific PCR amplification of cell-free DNA molecules originating from the specific genomic regions” in claims 28, fails to enable one skilled in the art to make and use the claimed invention using the application as a guide.
Instant specification [0514] recites “any of the methods described herein may be totally or partially performed with a computer system including one or more processors, which can be configured to perform the steps”, however, the state of prior art does not provide evidence of “a computer program” capable of performing a methylation-aware assay, comprising enrichment of cell-free DNA molecules, i) contacting the plurality of cell-free DNA molecules with hybridization probes, or ii ) performing methylation-specific PCR amplification of cell-free DNA molecules. As such, based on the evidence regarding the state of the prior art, the specification, at the time the application was filed, does not teach one skilled in the art how to make and/or use the full scope of the claimed invention without undue experimentation.
Claim Rejections - 35 USC § 101
35 U.S.C. 101 reads as follows:
Whoever invents or discovers any new and useful process, machine, manufacture, or composition of matter, or any new and useful improvement thereof, may obtain a patent therefor, subject to the conditions and requirements of this title.
Claims 1-10, 12, 21-35, and 37 are rejected under 35 U.S.C. 101 because the claimed invention is directed to an abstract idea without significantly more.
The Supreme Court has established a two-step framework for this analysis, wherein a claim does not satisfy § 101 if (1) it is “directed to” a patent-ineligible concept, i.e., a law of nature, natural phenomenon, or abstract idea, and (2), if so, the particular elements of the claim, considered “both individually and as an ordered combination,” do not add enough to “transform the nature of the claim into a patent-eligible application.” Elec. Power Grp., LLC v. Alstom S.A., 830 F.3d 1350, 1353 (Fed. Cir. 2016) (quoting Alice, 134 S. Ct. at 2355). Applicant is also directed to MPEP 2106.
Step 1: The instantly claimed invention (claim(s) 1, 29, and 30 being representative) is directed to a method and (claim 28 being representative) is directed to a system. Therefore, the instantly claimed invention falls into one of the four statutory categories. [Step 1: YES]
Step 2A: First it is determined in Prong One whether a claim recites a judicial exception, and if so, then it is determined in in Prong Two if the recited judicial exception is integrated into a practical application of that exception.
Step 2A, Prong 1: Under the MPEP § 2106.04, the Step 2A (Prong 1) analysis requires determining whether a claim recites an abstract idea, law of nature, or natural phenomenon.
Claim(s) 1-10, 12, 21-35, and 37 recite the following steps which fall under the mathematical concepts, mental processes, and/or certain methods of organizing human activity groupings of abstract ideas:
Claim 1, 29, and 33 recite analyzing a plurality of cell-free DNA molecules from the biological sample the limitation analyzing cell-free DNA molecules can be particularly performed in human mind because human mind is able to analyze data) by determining a location of the cell-free DNA molecule in a reference genome (the limitation determining a location can be particularly performed in human mind because human mind is able to map sequence reads to determine a location, for example, aligning sequence reads); determining whether the cell-free DNA molecule is methylated at one or more sites (the limitation determining methylation can be particularly performed in human mind, because human mind is able to determine methylation state based on the alignment/ mapping), the one or more sites of each of the plurality of cell-free DNA molecules providing a plurality of sites (the limitation providing plurality of sites can particularly performed in human mind because human mind is able to provide/give multiple sites based on the analysis); for each of the plurality of sites: determining a respective number of cell-free DNA molecules that are methylated at the site (the limitation determining methylated cell-free DNA can be particularly performed in human mind, because human mind is able to determine methylation state based on alignment data); calculating a first methylation level of a first chromosomal region based on the respective numbers of cell-free DNA molecules methylated at sites within the first chromosomal region (the limitation calculating a first methylation level involves calculating using mathematical methods, and as such, falls within mathematical concepts groupings of abstract idea).
Claims 29 and 30 further recite determining sequences of the enriched cell-free DNA molecules (mental process of determining based on the result of an analysis); for each site of the plurality of sites, determining a respective number of the cell- free DNA molecules at the site that are methylated (the limitation determining methylated cell-free DNA can be particularly performed in human mind, because human mind is able to determine methylation state based on alignment data).
Claims 6 and 7 recite determining a first classification of a level of cancer based on the first methylation level comprises (mental process of determining a classification based on the result of an analysis): comparing the first methylation level to a first cutoff value (mathematical process of comparing values/mathematical relationship); and determining the first classification of the level of cancer based on the comparison (mental process of determining a classification based on the result of an analysis).
Claim 8 recites identifying a type of cancer associated with the organism (mental process of identifying based on the result of an analysis).
Claim 12 recites comparing the first methylation level to the first cutoff value includes: determining a difference between the first methylation level and a reference methylation level (mathematical calculation/mathematical process); and comparing the difference to a threshold corresponding to the first cutoff value (mathematical relationship/mathematical process).
Claim 22 recites determining whether the CpG island is hypermethylated relative to a reference group of samples of other organisms by comparing a methylation level of the CpG island to a respective cutoff value (mathematical relationship of comparing values/mathematical process; mental process of determining based on the result of an analysis), thereby determining hypermethylated CpG islands (mental process of determining based on the result of an analysis); determining respective methylation densities for the hypermethylated CpG islands ()mental process of determining based on the result of an analysis; calculating a cumulative score from the respective methylation densities (mathematical calculation/mathematical process); and comparing the cumulative score to a cumulative cutoff value to determine the first classification ((mathematical relationship of comparing values/mathematical process).
Claim 23 recites determining whether a fractional concentration of tumor DNA in the biological sample is greater than a minimum value (mathematical relationship/mathematical process; mental process of determining based on the result of an analysis); and if the fractional concentration of tumor DNA is not greater than the minimum value, flagging the biological sample (mathematical relationship/mathematical process; mental process of flagging based on the result of an analysis).
Claim 31 recites comparing the first methylation level to a corresponding value determined from other organisms (mathematical relationship/mathematical process).
Claim 34 recites determining levels of one or more protein markers (mental process of determining based on a result of ana analysis).
Claims 9-10, 21, 24-27, 32-33, and 35 provide further information about the abstract ideas of comparing to a cut-off value, determining a first classification, analyzing plurality of sites, identifying type of cancer, and determining a level of markers.
Additionally, claims 1-10, 12, 21-35, and 37 recite a correlation between cell-free DNA of a biological sample and calculating a methylation level, and as such, falls into judicial exception of Laws of nature and natural phenomena. See MPEP 2106(b) I.
The identified claims recite a law of nature, a natural phenomenon (product of nature) and/or fall into one of the groups of abstract ideas of mathematical concepts, mental processes, and/or certain methods of organizing human activity for the reasons set forth above. See MPEP 2106.04 (a)(2) III and MPEP 2106.04 (b) I. Therefore, claims are directed to one or more judicial exception(s) and require further analysis in Prong Two. [Step 2A, Prong 1: YES]
Step 2A: Prong 2: Under the MPEP § 2106.04, the Step 2A, Prong 2 analysis requires identifying whether there are any additional elements recited in the claim beyond the judicial exception(s), and evaluating those additional elements to determine whether they integrate the exception into a practical application of the exception. This judicial exception is not integrated into a practical application for the following reasons.
The additional elements of claim(s) 1-10, 12, 21-35, and 37 include the following.
Claim 1 recites performing a methylation-aware assay comprising enrichment of cell-free DNA molecules originating from specific genomic regions; enrichment comprises: (i) contacting the plurality of cell-free DNA molecules with hybridization probes to enrich for cell-free DNA molecules; or (ii) performing methylation-specific PCR amplification of cell-free DNA molecules.
Claim 2 recites sequencing of at least 60,000 cell-free DNA molecules.
Claim 3 amplification of the cell-free DNA molecules prior to said sequencing.
Claim 4 recites treating the cell-free DNA molecules with sodium bisulfite prior to the enrichment.
Claim 5 recites treating the cell-free DNA molecules with sodium bisulfite is part of Tet-assisted bisulfite conversion or oxidative bisulfite sequencing for a detection of 5-hydroxymethylcytosine.
Claim 28 recites a non-transitory computer readable medium comprising a plurality of instructions that, when executed, control a computer system.
Claim 29 recites performing a methylation-aware assay comprising: (i) treating the plurality of cell-free DNA molecules with sodium bisulfite;(ii) contacting the plurality of cell-free DNA molecules with hybridization probes to enrich for cell-free DNA molecules originating from specific genomic regions.
Claim 30 recites performing a methylation-aware assay comprising: (i) contacting the plurality of cell-free DNA molecules with a protein that binds methylated DNA;(ii) contacting the plurality of cell-free DNA molecules with hybridization probes to enrich for cell-free DNA molecules.
Claim 37 recites the methylation- specific PCR comprises multiplex PCR.
The additional elements of a non-transitory computer readable medium comprising a plurality of instructions are generic computer components and/or processes. There are no limitations that indicate that the processor, input module, processing module, or output module in the computer-implemented system require anything other than generic computing systems. The courts have found the use of a computer or other machinery in its ordinary capacity for economic or other tasks (e.g., to receive, store, or transmit data) or simply adding a general-purpose computer or computer components after the fact to an abstract idea (e.g., a fundamental economic practice or mathematical equation) does not integrate a judicial exception into a practical application. See MPEP 2106.05(f).
Furthermore, the additional elements performing methylation- aware sequencing comprising enrichment of cell-free DNA molecules enrichment comprises: contacting the plurality of cell-free DNA molecules with hybridization probes to enrich for cell-free DNA molecules, sequencing, amplification, treatment with bisulfite prior to enrichment as part of Tet-assisted bisulfite conversion, contacting the plurality of cell-free DNA molecules with a protein that binds methylated DNA, performing methylation-specific PCR amplification of cell-free DNA molecules; treating the plurality of cell-free DNA molecules with sodium bisulfite, and the methylation- specific PCR comprises multiplex PCR equate to mere data gathering to provide data that is analyzed by the judicial exception. The courts have identified limitations that merely gather data as insignificant extra-solution activity that does not integrate the abstract idea into a practical application (see MPEP 2106.05(g)).
Therefore, the additionally recited elements amount to generic computer components and/or insignificant extra-solution activity and, as such, the claims as a whole do no integrate the abstract idea into practical application. See MPEP 2106.05(g). Thus, claims 1-10, 12, 21-35, and 37 are directed to an abstract idea. [Step 2A, Prong 2: NO]
Step 2B: In the second step it is determined whether the claimed subject matter includes additional elements that amount to significantly more than the judicial exception. An inventive concept cannot be furnished by an abstract idea itself. See MPEP § 2106.05.
The additional elements of claim(s) 1-10, 12, 21-35, and 37 include the following.
Claim 1 recites performing a methylation-aware assay comprising enrichment of cell-free DNA molecules; enrichment comprises: (i) contacting the plurality of cell-free DNA molecules with hybridization probes to enrich for cell-free DNA molecules; or (ii) performing methylation-specific PCR amplification of cell-free DNA molecules.
Claim 2 recites sequencing of at least 60,000 cell-free DNA molecules.
Claim 3 amplification of the cell-free DNA molecules prior to said sequencing.
Claim 4 recites treating the cell-free DNA molecules with sodium bisulfite prior to the enrichment.
Claim 5 recites treating the cell-free DNA molecules with sodium bisulfite is part of Tet-assisted bisulfite conversion or oxidative bisulfite sequencing for a detection of 5-hydroxymethylcytosine.
Claim 28 recites a non-transitory computer readable medium comprising a plurality of instructions that, when executed, control a computer system.
Claim 29 recites performing a methylation-aware assay comprising:(i) treating the plurality of cell-free DNA molecules with sodium bisulfite;(ii) contacting the plurality of cell-free DNA molecules with hybridization probes to enrich for cell-free DNA molecules originating from specific genomic regions.
Claim 30 recites performing a methylation-aware assay comprising:(i) contacting the plurality of cell-free DNA molecules with a protein that binds methylated DNA;(ii) contacting the plurality of cell-free DNA molecules with hybridization probes to enrich for cell-free DNA molecules.
Claim 37 recites the methylation- specific PCR comprises multiplex PCR.
The additional elements of a non-transitory computer readable medium comprising a plurality of instructions are conventional computer components and/or processes. The courts have found the use of a computer or other machinery in its ordinary capacity for economic or other tasks (e.g., to receive, store, or transmit data) or simply adding a general-purpose computer or computer components after the fact to an abstract idea (e.g., a fundamental economic practice or mathematical equation) does not provide significantly more. See Affinity Labs v. DirecTV, 838 F.3d 1253, 1262, 120 USPQ2d 1201, 1207 (Fed. Cir. 2016) (cellular telephone); TU Communications LLC v. AV Auto, LLC, 823 F.3d 607,613,118 USPQ2d 1744, 1748 (Fed. Cir. 2016) (computer server and telephone unit).
Furthermore, the additional elements of treating the cell-free DNA molecules with sodium bisulfite that is part of Tet-assisted bisulfite conversion or oxidative bisulfite sequencing for a detection of 5-hydroxymethylcytosine; and performing methylation-aware massively parallel sequencing amounts to conventional methods for performing sequencing to indicate methylation state. This position is supported by Krueger et al. (DNA methylome analysis using short bisulfite sequencing data, nature methods, VOL.9 NO.2, FEBRUARY 2012). Krueger et al. discloses that Bisulfite conversion of genomic DNA combined with next-generation sequencing (BS-seq) is widely used to measure the methylation state of a whole genome, the methylome, at single-base resolution (abstract). Krueger et al. further discloses that Bisulfite treatment of 5-hydroxymethylcytosine (5hmC) yields a similar intermediate to 5mC, meaning that BS-seq can be used to detect whether a position is (hydroxy-) methylated (p. 146, col. 1, para. 1).
Furthermore, the additional elements of performing methylation- aware assay comprising enrichment of cell-free DNA molecules enrichment comprises: performing methylation-specific PCR, sequencing, amplification, treatment with bisulfite as part of Tet-assisted bisulfite conversion or oxidative bisulfite sequencing are conventional processes of performing sequencing. This position is supported by Bryzgunova et al. (Methylation-Specific Sequencing of GSTP1 Gene Promoter in Circulating/Extracellular DNA from Blood and Urine of Healthy Donors and Prostate Cancer Patients, 16 September 2008, New York Academy of Sciences, pages: 222 – 225). Bryzgunova discloses that methylation-specific sequencing of the GSTP1 gene promoter in circulating cell-free DNA (cfDNA) from blood and urine can detect prostate cancer, identifying tumor-specific methylation patterns in body fluids, providing a potential noninvasive tool for diagnosis (abstract). Bryzgunova discloses obtaining samples; isolated cf-DNA was modified by sodium bisulfite (BioSilica), designing PCR primers, amplification, sequencing, for example, methylation-specific PCR (pg. 223, subsection: materials and methods; paras. 1-6).
Korshunova et al. (2008: Massively parallel bisulphite pyrosequencing reveals the molecular complexity of breast cancer-associated cytosine-methylation patterns obtained from tissue and serum DNA, 18:19-29) discloses an analysis of the DNA-methylation profiles of loci of four aberrantly methylated genomic regions at high frequency in correlation with breast cancer (CGI) in over 700,000 patient derived DNA fragments from cancer-free and breast carcinomas sera obtained via a massively parallel bisulfite sequencing (pg. 20, Col. 1).
Cortese et al. (Epigenetic markers of prostate cancer in plasma circulating DNA, 22 May 2012, Human Molecular Genetics, Volume 21, Issue 16, 15 August 2012, Pages 3619–3631) discloses machine-learning techniques to develop a multi-locus biomarker that correctly distinguished prostate cancer samples from unaffected controls using cirDNA (abstract). Cortese further discloses enrichment by hybridization probes purified enriched modified cirDNA was labeled using Cy3 (GE Healthcare, Baie d'Urfe, QC, Canada) and blood DNA reference pool was labeled using Cy5 (GE Healthcare) and co-hybridized to the HCGI12k microarrays (UHN, Toronto, ON, Canada) (pg. 3627, col. 2, para. 1).
Furthermore, the additional elements methylation- specific PCR comprises multiplex PCR is a conventional method of performing methylation-aware PCR. This position is supported by Egger et al. (DNA methylation testing and marker validation using PCR: diagnostic applications, Expert Rev. Mol. Diagn. 12(1), 75–92 (2012), Published online: 09 Jan 2014). Egger discloses methods of DNA methylation testing using PCR (abstract). Egger further discloses using multiplex-PCR for targeting genomic regions with known methylation status in normal and tumor DNA suitable for minimally invasive diagnosis, where small amounts of free circulating DNA can be tracked in different body fluids, such as blood, urine or sputum (pg. 77, col. 1, first para. and pg. 83, col. 2, para. 1). Additionally, E Bölke et al. (Methylated APC and GSTP1 genes in serum DNA correlate with the presence of circulating blood tumor cells and are associated with a more aggressive and advanced breast cancer disease, European Journal of Medical Research, Published: 26 July 2010, Volume 15, article number 277, (2010), pages 277-286). E Bölke discloses a method of validating tumor-specific epigenetic alterations in the cell-free DNA found in the peripheral blood of breast cancer patients and to assess whether a correlation exists between tumor-specific methylated DNA, CtC and the clinical status of patients diagnosed with breast cancer by carrying out multiplex PCR (pg. 278, col. 2, para. 1-pg. 279, col. 2, para. 2).
Kim et al. (2011: Deep sequencing reveals distinct patterns of DNA methylation in prostate cancer. Genome research 21.7: 1028-1041) mapped global CGI methylation patterns of prostate cancer, using MethylPlex-next-generation sequencing (M-NGS) and bisulfite conversion, to identify ~68,000 methylated regions per sample, especially of promoter regions for 2481 cancer-specific differentially methylated regions (DMRs) [Abstract, FIG 1, p1031 Col 2].
Varley et al. (2010: Bisulfite Patch PCR enables multiplexed sequencing of promoter methylation across cancer samples. Genome research, 20(9), 1279- 1287) demonstrates tumor-specific aberrant DNA methylation in colon and breast cancer with next generation sequencing/bisulfite techniques, obtaining almost 100K reads aligned to bisulfite treated reference sequences, analyzing 50+ differentially methylated loci (CGI) across a large number of samples (pg. 1281, Col. 1, FIG 2A).
Therefore, the additional element is not sufficient to amount to significantly more than the judicial exception.
Taken alone, the additional elements do not amount to significantly more than the above-identified judicial exception(s). Even when viewed as a combination, the additional elements fail to transform the exception into a patent-eligible application of that exception. Thus, the claims as a whole do not amount to significantly more than the exception itself. [Step 2B: NO]
Therefore, the instantly rejected claims are not drawn to eligible subject matter as they are directed to an abstract idea without significantly more.
Claim Rejections - 35 USC § 103
In the event the determination of the status of the application as subject to AIA 35 U.S.C. 102 and 103 (or as subject to pre-AIA 35 U.S.C. 102 and 103) is incorrect, any correction of the statutory basis (i.e., changing from AIA to pre-AIA ) for the rejection will not be considered a new ground of rejection if the prior art relied upon, and the rationale supporting the rejection, would be the same under either status.
The following is a quotation of 35 U.S.C. 103 which forms the basis for all obviousness rejections set forth in this Office action:
A patent for a claimed invention may not be obtained, notwithstanding that the claimed invention is not identically disclosed as set forth in section 102, if the differences between the claimed invention and the prior art are such that the claimed invention as a whole would have been obvious before the effective filing date of the claimed invention to a person having ordinary skill in the art to which the claimed invention pertains. Patentability shall not be negated by the manner in which the invention was made.
The factual inquiries for establishing a background for determining obviousness under 35 U.S.C. 103 are summarized as follows:
1. Determining the scope and contents of the prior art.
2. Ascertaining the differences between the prior art and the claims at issue.
3. Resolving the level of ordinary skill in the pertinent art.
4. Considering objective evidence present in the application indicating obviousness or nonobviousness.
This application currently names joint inventors. In considering patentability of the claims the examiner presumes that the subject matter of the various claims was commonly owned as of the effective filing date of the claimed invention(s) absent any evidence to the contrary. Applicant is advised of the obligation under 37 CFR 1.56 to point out the inventor and effective filing dates of each claim that was not commonly owned as of the effective filing date of the later invention in order for the examiner to consider the applicability of 35 U.S.C. 102(b)(2)(C) for any potential 35 U.S.C. 102(a)(2) prior art against the later invention.
Claims 1-4, 25-28, 29-30, and 37 are rejected under 35 U.S.C. 103 as being unpatentable over Korshunova et al. (2008: Massively parallel bisulphite pyrosequencing reveals the molecular complexity of breast cancer-associated cytosine-methylation patterns obtained from tissue and serum DNA, 18:19-29), in view of Tynan et al. (US20140093873A1).
Regarding claims 1, 29, and 30, Korshunova discloses a method of detecting a chromosomal abnormality from a biological sample of a subject, the biological sample including cell-free DNA comprising a mixture of cell-free DNA originating from a first tissue and from a second tissue, the method comprising (a method of DNA methylation-based cancer-detection tests by analysing cell-free DNA in both tumor and cancer-free tissues (abstract)): analyzing a plurality of cell-free DNA molecules from the biological sample (abstract), wherein analyzing a cell-free DNA molecule includes: determining a location of the cell-free DNA molecule in a reference genome (p. 20, col.2, para. 3, mapping the results to particular patient samples using MethylMapper procedure); determining whether the cell-free DNA molecule is methylated at one or more sites, the one or more sites of each of the plurality of cell-free DNA molecules providing a plurality of sites for each of the plurality of sites: determining a respective number of cell-free DNA molecules that are methylated at the site (p. 20, col. 1, last para., DNA-methylation landscape present in just over 700,000 patient derived DNA fragments from cancer-free breast tissue, infiltrating ductal breast carcinomas, and sera obtained from a collection of 50 patients using a massively parallel bisulphite sequencing strategy. The targeted loci in the investigation were four genomic regions that we have; recently identified as aberrantly methylated at high frequency in correlation with breast cancer to provides information regarding the epigenetic pattern normally observed in DNA circulating within the bloodstream); calculating a first methylation level using the respective numbers of cell-free DNA molecules methylated at sites (p. 23, col. 2, para. 1, The cytosine-methylation topography within the samples was analyzed according to two criteria: the methylation pattern of each molecule and methylation density or the percent of the methylated residues from the total number of residues sequenced per molecule. The average methylation density of a region is the mean methylation occupancy per CG across a region (i.e., amplicon). Within a region, the average methylation occupancy was calculated for each CG as a percent of the methylated C's from the total number of molecules sequenced at that position).
Further regarding claims 1, 29, and 30 Korshunova discloses that the locus-specific tag-bearing PCR products were enriched using coded primers, and amplified from bisulphate-modified DNA, purified, quantified individually, mixed in equimolar amounts, and sequenced using the 454-pyrosequencing platform (pg. 20, col. 2, last para.).
Tynan discloses non-invasive methylation-based enrichment of circulating DNA methods that utilize genomic regions that are differentially methylated between a mother and her fetus to separate, isolate or enrich fetal nucleic acid from a maternal sample non-invasively.
Tynan further discloses treating sample with a reagent that differentially modifies methylated and unmethylated DNA. For example, the reagent may comprise bisulfite; or the reagent may comprise one or more enzymes that preferentially cleave methylated DNA; or the reagent may comprise one or more enzymes that preferentially cleave unmethylated DNA (for example, methylation-aware assay) [0021].
Tynan discloses enriching a sample nucleic acid for a plurality of polymorphic nucleic acid targets (claim 1), where enriching comprises methylation sensitive capture, for example using, MBD2-Fc fragment; bisulfite conversion methods, for example, MSP (methylation-sensitive PCR), COBRA, methylation-sensitive single nucleotide primer extension (Ms-SNuPE) or Sequenom MassCLEAVE™ technology; and the use of methylation sensitive restriction enzymes [0031]. Tynan further discloses MSP (methylation-specific PCR) allows for assessing the methylation status and furter discloses that MSP requires only small quantities of DNA, is sensitive to 0.1% methylated alleles of a given CpG island locus, and can be performed on DNA extracted from paraffin-embedded samples. Typical reagents (e.g., as might be found in a typical MSP-based kit) for MSP analysis may include, but are not limited to: methylated and unmethylated PCR primers for specific gene (or methylation-altered DNA sequence or CpG island), optimized PCR buffers and deoxynucleotides, and specific probes [0225-0226] [0262-0282]. Tynan further discloses Nimblegen sequence capture system (Roche NimbleGen, Madison, Wis.); Illumina BEADARRAY platform (Illumina, San Diego, Calif.) (for example, targeted hybridization probe); Affymetrix GENECHIP platform (Affymetrix, Santa Clara, Calif.); Agilent SureSelect Target Enrichment System (Agilent Technologies, Santa Clara, Calif.) (for example, targeted hybridization probe) [0278].
Further regarding claim 29, Korshunova discloses four biological samples (two from cancer patients and two from cancer-free individuals) were bisulphite converted, amplified, and sequenced in two independent experiments incorporating a position-specific BLAST requirement using barcoded primers to amplify cfDNA (pg.22, col.1 last para.); The targeted loci in the investigation were four genomic regions that we have recently identified as aberrantly methylated at high frequency in correlation with breast cancer (pg. 20, col. 1, last para.; Figure 1). Tynan discloses preparing the cfDNA comprises a hybridization process, a capture process, or an amplification process [0011]; Hyper- and hypomethylated nucleic acid sequences of the technology herein are identified [0014-0016]; see also quantitative methylation assay to determine methylation levels [0218].
Further regarding claim 30, Tynan discloses that MBD-FC was used to capture the methylated fraction of each DNA sample. See FIGS. 1-3. The two tissue fractions were labeled with different fluorescent dyes and hybridized to an Agilent® CpG Island microarray to identify differentially methylated region [0384] [0368] [0269].
Regarding claim 2, Korshunova discloses analysis of the DNA-methylation landscape present in just over 700,000 patient derived DNA fragments from cancer-free breast tissue, infiltrating ductal breast carcinomas, and sera obtained from a collection of 50 patients using a massively parallel bisulphite sequencing strategy (pg. 20, col. 1, last para.).
Tynan discloses that enriched (e.g., amplified) polymorphic nucleic acid targets are sequenced by a sequencing process [0258]. Tynan further discloses that any sequencing method suitable for conducting methods described herein can be utilized; a massively parallel sequencing, which sequence millions of cfDNA; High-throughput sequencing technologies include, for example, sequencing-by-synthesis with reversible dye terminators, sequencing by oligonucleotide probe ligation, pyrosequencing and real time sequencing [0336]; reading on limitations of performing the methylation-aware assay comprises sequencing of at least 60,000 cell-free DNA molecules.
Regarding claim 3, Tynan discloses PCR amplification of the bisulfite converted DNA [0218]; reading on limitations of performing the methylation-aware assay further comprises amplification of the cell-free DNA molecules prior to said sequencing.
Regarding claim 4, Tynan discloses treating the cell-free DNA molecules with sodium bisulfite [0199] [0218] [0219] [0221] [0223]; reading on limitations of treating the cell-free DNA molecules with sodium bisulfite prior to the enrichment.
Regarding claim 25, Korshunova discloses that the genomic loci under the study are chromosomes (Figure 1). Tynan discloses detecting the presence or absence of fetal aneuploidy, the amount of fetal nucleic acid may be determined at multiple loci on one or more target chromosomes (e.g., chromosomes 13, 18 or 21) and on one or more reference chromosomes [0041] [0094] [0267] [0371].
Regarding claim 26, Tynan discloses that two or more independent loci in the target chromosome are assayed [0067] [0094]; reading on limitations of wherein the plurality of sites are from disjointed regions separated from each other.
Regarding claim 27, Tynan discloses that for identification of differentially methylated regions, a novel approach was used to capture methylated DNA. This approach uses a protein, in which the methyl binding domain of MBD2 is fused to the Fc fragment of an antibody (MBD-FC) [0211]; reading on limitations of the methylation-aware assay further comprises contacting the cell-free DNA molecules with a protein that binds methylated DNA.
Regarding claim 28, Tynan discloses a computer readable medium (e.g., optical and/or magnetic storage or transmission medium, floppy disk, hard disk, random access memory, computer processing unit [0044]; reading on limitations of a non-transitory computer readable medium comprising a plurality of instructions that, when executed, control a computer system to perform the method of claim 1.
Regarding claim 37, Tynan discloses that the amplification method is multiplex PCR [0273]; reading on limitations of the methylation- specific PCR comprises multiplex PCR.
In KSR Int 'l v. Teleflex, the Supreme Court, in rejecting the rigid application of the teaching, suggestion, and motivation test by the Federal Circuit, indicated that “The principles underlying [earlier] cases are instructive when the question is whether a patent claiming the combination of elements of prior art is obvious. When a work is available in one field of endeavor, design incentives and other market forces can prompt variations of it, either in the same field or a different one. If a person of ordinary skill can implement a predictable variation, § 103 likely bars its patentability.” KSR Int'l v. Teleflex lnc., 127 S. Ct. 1727, 1740 (2007).
Applying the KSR standard to Korshunova and Tynan, the examiner concludes that the combination of Korshunova and Tynan represents the use of known techniques to improve similar methods. Both Korshunova and Tynan are directed to methylation profiling of cell-free DNA. Korshunova discloses that the locus-specific tag-bearing PCR products were enriched using coded primers, and amplified from bisulphate-modified DNA, purified, quantified individually, mixed in equimolar amounts, and sequenced using the 454-pyrosequencing platform. In the same field of research, Tynan provided the known technique of using targeted hybridization probes or methylation-specific PCR for the purpose of enriching the samples. Combining the methylation profiling of Korshunova comprising faster primer-based enrichment with targeted enrichment of Tynan would have allowed for greater genomic coverage, higher sensitivity to mutations, and better handling the methylated DNA. One ordinary skilled in the art before he effective filing data of the claimed invention would have had a reasonable expectation of success at combining the method of Korshunova and Tynan. This combination would have been expected to have provided a more specific methylation profiling. Therefore, the invention would have been prima facie obvious to one of skill in the art before the effective filing date of the claimed invention, absent evidence to the contrary.
Claim 5 is rejected under 35 U.S.C. 103 as being unpatentable over Korshunova et al. (2008: Massively parallel bisulphite pyrosequencing reveals the molecular complexity of breast cancer-associated cytosine-methylation patterns obtained from tissue and serum DNA, 18:19-29), in view of Tynan et al. (US20140093873A1), as applied to claims 1-4, 25-28, 29-30, and 37 above, and further in view of Booth et al. (Quantitative Sequencing of 5-Methylcytosine and 5-Hydroxymethylcytosine at Single-Base Resolution, 18 MAY 2012 VOL 336 SCIENCE, p. 934-937).
Claim 5 depend on claim 4 and 1. The limitations of claims 1 and 4 have been taught in the above rejection.
Regarding claim 5, Korshunova et al. discloses Massively parallel bisulphite pyrosequencing (abstract).
Tynan discloses nucleic acid may be treated with (i) alkylating agents such as methylnitrosourea that generate several alkylated bases, including N3-methyladenine and N3-methylguanine, which are recognized and cleaved by alkyl purine DNA-glycosylase; (ii) sodium bisulfite, which causes deamination of cytosine residues in DNA to form uracil residues that can be cleaved by uracil N-glycosylase; and (iii) a chemical agent that converts guanine to its oxidized form, 8-hydroxyguanine, which can be cleaved by formamidopyrimidine DNA N-glycosylase [0199].
Booth discloses oxidative bisulfite sequencing (oxBS-Seq), the first method for quantitative mapping of 5hmC in genomic DNA (abstract).
It would have been prima facie obvious to one of ordinary skill in the art, before the effective filing date of the claimed invention, to have modified the method of Korshunova and Tynan to have treated the DNA molecules with the sodium bisulfite as part of Tet-assisted bisulfite conversion or oxidative bisulfite sequencing for a detection of 5-hydroxymethylcytosine, as shown by Booth et al. (abstract). There would be a reasonable expectation of success in combining the technique of Booth to the method of Korshunova and Tynan because they all use bisulfite sequencing to reveal methylation patterns.
Claims 6-8, 21-22, and 31-35 are rejected under 35 U.S.C. 103 as being unpatentable over Korshunova et al. (2008: Massively parallel bisulphite pyrosequencing reveals the molecular complexity of breast cancer-associated cytosine-methylation patterns obtained from tissue and serum DNA, 18:19-29), in view of Tynan et al. (US20140093873A1), as applied to claims 1-4, 25-28, 29-30, and 37 above, and further in view of Melnikov et al. (US20080261217A1).
Claims 6 and 7 depend on claim 1. Limitations of claim 1 has been taught in the above rejections.
Regarding claims 6 and 7, Korshunova discloses analyzing cytosine-methylation topography within the samples; the methylation pattern of each molecule and methylation density (or the percent of the methylated residues from the total number of residues sequenced per molecule). The average methylation density of a region is the mean methylation occupancy per CG across a region; allowing characterization of the methylation level of the molecular population without analyzing prevalence of any molecular configuration; The goal of the analysis was to identify either a configuration or a regional methylation density where the relative abundance of each may be diagnostic of breast cancer (pg. 23, col. 2, para. 1). Korshunova further discloses performing discriminative analysis for classification of tumor (pg. 2, col. 2, last para.); Tynan discloses assigning classification (normal vs diseased) to sample [0161]; determining the methylation levels [0369]; genomic regions were classified as being not differentially methylated when the group showed less than eight samples with a p value <0.01 and less than six samples with a p value <0.001[0390].
Further regarding claims 6 and 7, Melnikov discloses that the methylation profile of the subject may be compared to a standard methylation profile (e.g., a standard methylation profile for non-cancerous samples, a standard methylation profile for cancerous samples, or both) [0020] [0046]. Melnikov further discloses using thresholds for methylated fragments defining methylated and unmethylated calls for diagnosis of cancer [0090].
Regarding claim 8, Melnikov discloses methods of identifying methylation patterns in genes associated with specific cancers (abstract). Melnikov further discloses a) reacting isolated genomic DNA from the subject and a methylation-sensitive restriction enzyme; wherein the genomic DNA comprises a plurality of promoters from different genes, and the enzyme cleaves unmethylated CpG sequences in the promoters and does not cleave methylated CpG sequences in the promoters; (b) contacting the genomic DNA thus reacted and a plurality of pairs of specific primers in an amplification mixture, the pairs of specific primers being configured to hybridize to the genomic DNA and to amplify a plurality of different promoters through a region comprising an uncleaved CpG sequence; (c) reacting the amplification mixture; (d) detecting one or more amplified promoters in the reacted amplification mixture or the absence thereof, thereby diagnosing cancer in the subject selected from the group consisting of ovarian cancer, lung cancer, prostate cancer, pancreatic cancer, and colon cancer (claim 1); a cell-free assay is provided in which a cancer marker gene, protein or biologically active portion thereof is contacted with a test compound and the ability of the test compound to bind to the cancer marker gene, protein or biologically active portion thereof is evaluated [0116].
Melnikov further discloses distinguishing normal from cancer status and cancer classification [0262-0268]; reading on limitations of the first classification indicates that cancer exists for the organism, the method further comprising identifying a type of cancer associated with the organism.
Regarding claims 21 and 22, Korshunova discloses variety of molecular methylation patterns recovered from each run including CpG sites (pg. 23, col. 1, para. 1; Table 2; Figure 1). Tynan discloses separating fetal and maternal nucleic acid based on the methylation status of a CpG-containing genomic sequence in the sample [0009]; assays allowing for determination of the methylation state of one or a plurality of CpG islands within a DNA sequence [0216]; PCR amplification of the bisulfite converted DNA is then performed using primers specific for the interested CpG islands, followed by restriction endonuclease digestion, gel electrophoresis, and detection using specific, labeled hybridization probes. Methylation levels in the original DNA sample are represented by the relative amounts of digested and undigested PCR product in a linearly quantitative fashion across a wide spectrum of DNA methylation levels [0218] [0457]. Tynan further discloses CpG site quantification using Ms-SNuPE technique, where Methylation densities are calculated providing a quantitative score (a percentage of methylated vs. unmethylated cytosines) [0223]; see also, cutoff values of a set of standards for determining a CpG island: it must be at least 400 nucleotides in length, has a greater than 50% GC content, and an OCF/ECF ratio greater than 0.6. Others [0128].
Melnikov discloses providing differential methylation of CpG islands (claim 1) [0092] [0094]; demonstration of increased frequency of CpG methylation over normal [0223].
Regarding claim 31, Melnikov discloses Predicted status for each sample (e.g. pCancer, pADH, pNormal, etc) was compared with its true status (Cancer, ADH, Normal, etc). Intersection of predicted and true status for each type of cancer shows the sensitivity (e.g. 72.39% of Cancer samples are correctly identified, so the sensitivity of cancer classifier is 72.39%), while intersection of predicted and true status of Normals indicates the specificity of the classifier (e.g. 74.74% of Normal samples are correctly identified by the cancer classifier, so its specificity is 74.74%) [0264]; The fraction of U calls for each tissue type is shown with p-values from Fisher's Exact Test for differential methylation on 2×2 tables for all pairwise comparisons [0267]; reading on limitations of identifying the type of cancer associated with the organism comprises comparing the first methylation level to a corresponding value determined from other organisms, wherein at least two of the other organisms are identified as having different types of cancer.
Regarding claims 32 and 33, Melnikov discloses that MethDet can be considered as the first-line test in combination with TVUS or other imaging techniques; While the accuracy of developed biomarkers needs additional refinement, even at this time the blood-based biomarker can be useful as a first-line screening tool in combination with imaging techniques [0333-0334]; radiological screening techniques include (mammography, ultrasonography, computed tomography, magnetic resonance imaging) [0004]; reading on limitations of identifying the type of cancer associated with the organism comprises radiological and/or imaging investigation, and wherein the imaging comprises computed tomography, magnetic resonance imaging, or positron emission tomography.
Regarding claims 34 and 35, Tynan discloses that Nucleic acid species with a different methylation status can be differentiated by a methylation-specific binding protein such as MBD-Fc [0029]. Melnikov discloses measuring the levels of CA125 [02910293]; reading on limitations of determining levels of one or more protein markers, wherein the one or more protein markers is selected from the group consisting of prostate specific antigen, carcinoembryonic antigen, alpha fetoprotein, CA125 and CA19-9.
It would have been prima facie obvious to one of ordinary skill in the art, before the effective filing date of the claimed invention, to have modified the method of Korshunova and Tynan to have used their methylation profiling method to classify abnormalities such as cancer, as shown by Melnikov. There would be a reasonable expectation of success in combining the method of Melnikov with the method of Korshunova and Tynan because they all use methylation profiling to identify one or more abnormalities.
Claims 8 and 31-35 are rejected under 35 U.S.C. 103 as being unpatentable over Korshunova et al. (2008: Massively parallel bisulphite pyrosequencing reveals the molecular complexity of breast cancer-associated cytosine-methylation patterns obtained from tissue and serum DNA, 18:19-29), in view of Tynan et al. (US20140093873A1), as applied to claims 1-4, 25-28, 29-30, and 37 above, and further in view of Melnikov et al. (US20080261217A1), as applied to claims 6-8, 21-22, and 31-35 above, and further in view of Fackler et al. (Quantitative Multiplex Methylation-Specific PCR Assay for the Detection of Promoter Hypermethylation in Multiple Genes in Breast Cancer, CANCER RESEARCH 64, 4442–4452, July 1, 2004).
Claims 9 depend on claims 1 and 7. Limitations of claim 1 and 7 have been taught in the above rejections.
Regarding claim 9, Freckle et al. discloses that the cutoff value is determined from other subjects not having cancer (Table 5, 6; p. 4448, cols. 1&2, The cumulative methylation profiles of 9 normal mammoplasty samples were compared with those of 19 invasive carcinomas).
Regarding claim 10, Fackler et al. discloses that the cutoff value is a specified distance from a reference methylation level established from another biological sample obtained from a healthy subject or a chromosomal region that does not have the abnormality (pg 4447, col 2, paras 1 and 2, determining whether a CpG island is hypermethylated by using the Mann-Whitney test on the untransformed data; Figure 7, Cumulative promoter hypermethylation of RASSF1A, TWIST, Cyclin D2, and HIN1 in adjacent normal and malignant breast tissues).
Regarding claim 12, Fackler et al. discloses comparing the first methylation level to the cutoff value includes: determining a difference between the first methylation level and a reference methylation level; and comparing the difference to a threshold corresponding to the cutoff value (pg 4447, col 2, paras 1 & 2) the differences in the medians were highly significant for all genes tested… We chose to establish a cutoff (% M) for each gene at approximately the 10th percentile of the population, such that _90% of normal breast tissues would be at or below the cutoff. Using cutoffs of 2% M for RASSF1A and HIN1, 0.5% M for TWIST, and 0.2% M for Cyclin D2 in normal tissues, we considered values above the cutoffs “positive” for hypermethylation.
It would have been prima facie obvious to one of ordinary skill in the art, before the effective filing date of the claimed invention, to have modified the method of Korshunova, Tynan, and Melnikov to have used the cutoff values, as shown by Fackler et al. (Para 4; pg 4447, col 1, para 2 to pg 4449, col 2, para 1; Figures 5-8; Tables 5-7) to distinguish between different states. There would be a reasonable expectation of success in combining the technique of Fackler et al. to the method of Korshunova, Tynan, and Melnikov because they are all in the field of detecting hypermethylation in one or more abnormalities.
Claims 23-24 are rejected under 35 U.S.C. 103 as being unpatentable over Korshunova et al. (2008: Massively parallel bisulphite pyrosequencing reveals the molecular complexity of breast cancer-associated cytosine-methylation patterns obtained from tissue and serum DNA, 18:19-29), in view of Tynan et al. (US20140093873A1), as applied to claims 1-4, 25-28, 29-30, and 37 above, and further in view of Wong et al. (Quantitative Analysis of Tumor-derived Methylated p16INK4a Sequences in Plasma, Serum, and Blood Cells of Hepatocellular Carcinoma Patients, clinical Cancer Research, (2003) 9 (3): 1047–1052. 03/01/2003).
Claims 23-24 depend on claim 1. Limitations of claim 1 have been taught in the above rejections.
Regarding claims 23 and 24, Tynan discloses determining fetal fraction based on the allele frequency mean; the fetal genotype at one or more informative polymorphic nucleic acid targets is heterozygous; the fetal genotype at one or more informative polymorphic nucleic acid targets is homozygous; fetal fraction is determined with a coefficient of variance (CV) of 0.20 or less; fetal fraction is determined with a coefficient of variance (CV) of 0.10 or less, and sometimes fetal fraction is determined with a coefficient of variance (CV) of 0.05 or less [0073] [0246].
Wong discloses method of quantifying methylated sequences and determining the fractional concentrations of circulating tumor DNA in plasma, serum, and peripheral blood cells (abstract). Wong further discloses that the fractional concentration of circulating tumor-derived DNA in plasma, the proportion of bisulfite converted unmethylated and methylated p16INK4a sequences that consisted of tumor-derived methylated p16INK4a sequences, was calculated for each methylation-positive plasma sample. For the 9 preoperative plasma samples, the p16INK4a methylation indices were 0.2, 0.4, 0.6, 21.3, 35, 46.7, 73.1, 85.3, and 100% (median methylation index = 35%; n = 9; Table 2). On the other hand, the p16INK4a methylation indices on the 8 postoperative plasma samples (for example, a reference methylation level) were 0.3, 0.3, 0.4, 1.6, 4.5, 11.2, 28, and 71.5% (median methylation index = 3.05%; n = 8; Table 2), corresponding to 10, 13, 32, 34, 34, 60, 77, and 103 genome-equivalents of methylated sequences/ml (median quantity = 34 genome-equivalents/ml); reading on limitations of determining whether a fractional concentration of tumor DNA in the biological sample is greater than a minimum value; and if the fractional concentration of tumor DNA is not greater than the minimum value, flagging the biological sample.
It would have been prima facie obvious to one of ordinary skill in the art, before the effective filing date of the claimed invention, to have modified the method of Korshunova and Tynan to have used tumor fraction/fractional concentration of tumor DNA, as shown by Wong, to distinguish cancer-derived DNA from healthy blood cell DNA. There would be a reasonable expectation of success in combining the technique of Wong to the method of Korshunova and Tynan because they are all in the field of detecting hypermethylation in one or more abnormalities.
Double Patenting
The nonstatutory double patenting rejection is based on a judicially created doctrine grounded in public policy (a policy reflected in the statute) so as to prevent the unjustified or improper timewise extension of the “right to exclude” granted by a patent and to prevent possible harassment by multiple assignees. A nonstatutory double patenting rejection is appropriate where the conflicting claims are not identical, but at least one examined application claim is not patentably distinct from the reference claim(s) because the examined application claim is either anticipated by, or would have been obvious over, the reference claim(s). See, e.g., In re Berg, 140 F.3d 1428, 46 USPQ2d 1226 (Fed. Cir. 1998); In re Goodman, 11 F.3d 1046, 29 USPQ2d 2010 (Fed. Cir. 1993); In re Longi, 759 F.2d 887, 225 USPQ 645 (Fed. Cir. 1985); In re Van Ornum, 686 F.2d 937, 214 USPQ 761 (CCPA 1982); In re Vogel, 422 F.2d 438, 164 USPQ 619 (CCPA 1970); In re Thorington, 418 F.2d 528, 163 USPQ 644 (CCPA 1969).
A timely filed terminal disclaimer in compliance with 37 CFR 1.321(c) or 1.321(d) may be used to overcome an actual or provisional rejection based on nonstatutory double patenting provided the reference application or patent either is shown to be commonly owned with the examined application, or claims an invention made as a result of activities undertaken within the scope of a joint research agreement. See MPEP § 717.02 for applications subject to examination under the first inventor to file provisions of the AIA as explained in MPEP § 2159. See MPEP § 2146 et seq. for applications not subject to examination under the first inventor to file provisions of the AIA . A terminal disclaimer must be signed in compliance with 37 CFR 1.321(b).
The filing of a terminal disclaimer by itself is not a complete reply to a nonstatutory double patenting (NSDP) rejection. A complete reply requires that the terminal disclaimer be accompanied by a reply requesting reconsideration of the prior Office action. Even where the NSDP rejection is provisional the reply must be complete. See MPEP § 804, subsection I.B.1. For a reply to a non-final Office action, see 37 CFR 1.111(a). For a reply to final Office action, see 37 CFR 1.113(c). A request for reconsideration while not provided for in 37 CFR 1.113(c) may be filed after final for consideration. See MPEP §§ 706.07(e) and 714.13.
The USPTO Internet website contains terminal disclaimer forms which may be used. Please visit www.uspto.gov/patent/patents-forms. The actual filing date of the application in which the form is filed determines what form (e.g., PTO/SB/25, PTO/SB/26, PTO/AIA /25, or PTO/AIA /26) should be used. A web-based eTerminal Disclaimer may be filled out completely online using web-screens. An eTerminal Disclaimer that meets all requirements is auto-processed and approved immediately upon submission. For more information about eTerminal Disclaimers, refer to www.uspto.gov/patents/apply/applying-online/eterminal-disclaimer.
Claims 1, 2, 4-7, 8-10, 12, 21-22, 25, 27-35, and 37 are rejected on the ground of nonstatutory double patenting as being unpatentable over claims 1, 7, 22, 23, 25-27, 29, and 33 of U.S. Patent No. 12,518,854 in view of Korshunova et al. (2008: Massively parallel bisulphite pyrosequencing reveals the molecular complexity of breast cancer-associated cytosine-methylation patterns obtained from tissue and serum DNA, 18:19-29), in view of Tynan et al. (US20140093873A1), and further in view of Melnikov et al. (US20080261217A1). Although the claims at issue are not identical, they are not patentably distinct from each other.
Regarding instant claims 1, 2, 29, and 30, reference claims 1 and 22 recite method of detecting an abnormality in a first tissue for a first chromosomal region from a biological sample of a subject, the biological sample including cell-free DNA comprising a mixture of cell-free DNA originating from the first tissue and from a second tissue, the method comprising: analyzing a plurality of cell-free DNA molecules from the biological sample, wherein analyzing a cell-free DNA molecule includes: determining a location of the cell-free DNA molecule in a reference genome; determining whether the cell-free DNA molecule is methylated at one or more sites, the one or more sites of each of the plurality of cell-free DNA molecules providing a plurality of sites, wherein determining whether the cell-free DNA molecule is methylated at the one or more sites comprises performing methylation-aware sequencing, wherein the plurality ofcell-free DNA molecules comprise at least 60,000;for each of the plurality of sites: determining a respective number of the at least 60,000 cell-free DNA molecules that are methylated at the site; calculating a first methylation level of the first chromosomal region based on the respective numbers of the at least 60,000 cell-free DNA molecules methylated at sites within the first chromosomal region; comparing the first methylation level to a cutoff value; and determining a classification of the abnormality in the first tissue for the first chromosomal region based on the comparison; treating the cell-free DNA molecules with sodium bisulfite;
Regarding instant claims 4 and 5, reference claims 22 and 23 recites performing methylation-aware sequencing includes: treating the cell-free DNA molecules with sodium bisulfite; and performing sequencing of the treated cell-free DNA molecules; and wherein treating the cell-free DNA molecules with the sodium bisulfite is part of Tet-assisted bisulfite conversion or oxidative bisulfite sequencing for a detection of 5-hydroxymethylcytosine.
Regarding instant claim 6, reference claims 1 and 7 recites determining a classification of the abnormality in the first tissue for the first chromosomal region based on the comparison; wherein the abnormality is cancer.
Regarding instant claim 7, reference claims 1 and 7 recite comparing the first methylation level to a cutoff value; and determining a classification of the abnormality in the first tissue for the first chromosomal region based on the comparison; wherein the abnormality is cancer.
Regarding instant claims 9 and 10, reference claims 26 and 27 recite wherein the cutoff value is a specified distance from a reference methylation level established from another biological sample obtained from a healthy subject or a chromosomal region that does not have the abnormality; and the specified distance is a specified number of standard deviations from the reference methylation level.
Regarding instant claim 12, reference claim 29 recites wherein comparing the first methylation level to the cutoff value includes: determining a difference between the first methylation level and a reference methylation level; and comparing the difference to a threshold corresponding to the cutoff value.
Regarding instant claims 21 and 22, reference claim 25 recites wherein the plurality of sites are CpG sites.
Regarding instant claim 25, reference claim 1 recites the plurality of sites are on a plurality of chromosomes.
Regarding instant claim 28, reference claim 33 recites non-transitory computer readable medium comprising a plurality of instructions that, when executed, control a computer system to perform the method of claim 1.
Reference claims do not disclose enrichment by targeted hybridization probes or methylation-specific PCR, amplification prior to sequencing, contacting CFDNA with a protein, identifying a type of cancer, a protein, determining hypermethylation of CpG islands.
Korshunova discloses that the locus-specific tag-bearing PCR products were enriched using coded primers, and amplified from bisulphate-modified DNA, purified, quantified individually, mixed in equimolar amounts, and sequenced using the 454-pyrosequencing platform (pg. 20, col. 2, last para.).
Tynan discloses non-invasive methylation-based enrichment of circulating DNA methods that utilize genomic regions that are differentially methylated between a mother and her fetus to separate, isolate or enrich fetal nucleic acid from a maternal sample non-invasively.
Tynan further discloses treating sample with a reagent that differentially modifies methylated and unmethylated DNA. For example, the reagent may comprise bisulfite; or the reagent may comprise one or more enzymes that preferentially cleave methylated DNA; or the reagent may comprise one or more enzymes that preferentially cleave unmethylated DNA (for example, methylation-aware assay) [0021].
Tynan discloses enriching a sample nucleic acid for a plurality of polymorphic nucleic acid targets (claim 1), where enriching comprises methylation sensitive capture, for example using, MBD2-Fc fragment; bisulfite conversion methods, for example, MSP (methylation-sensitive PCR), COBRA, methylation-sensitive single nucleotide primer extension (Ms-SNuPE) or Sequenom MassCLEAVE™ technology; and the use of methylation sensitive restriction enzymes [0031]. Tynan further discloses MSP (methylation-specific PCR) allows for assessing the methylation status and further discloses that MSP requires only small quantities of DNA, is sensitive to 0.1% methylated alleles of a given CpG island locus, and can be performed on DNA extracted from paraffin-embedded samples. Typical reagents (e.g., as might be found in a typical MSP-based kit) for MSP analysis may include, but are not limited to: methylated and unmethylated PCR primers for specific gene (or methylation-altered DNA sequence or CpG island), optimized PCR buffers and deoxynucleotides, and specific probes [0225-0226] [0262-0282]. Tynan further discloses Nimblegen sequence capture system (Roche NimbleGen, Madison, Wis.); Illumina BEADARRAY platform (Illumina, San Diego, Calif.) (for example, targeted hybridization probe); Affymetrix GENECHIP platform (Affymetrix, Santa Clara, Calif.); Agilent SureSelect Target Enrichment System (Agilent Technologies, Santa Clara, Calif.) (for example, targeted hybridization probe) [0278].
Further regarding claim 29, Korshunova discloses treating the plurality of cell-free DNA molecules with sodium bisulfite (pg.22, col.1 last para.); Tynan discloses preparing the cfDNA comprises a hybridization process, a capture process, or an amplification process [0011]; Hyper- and hypomethylated nucleic acid sequences of the technology herein are identified [0014-0016]; see also quantitative methylation assay to determine methylation levels [0218].
Further regarding claim 30, Tynan discloses that MBD-FC was used to capture the methylated fraction of each DNA sample. See FIGS. 1-3. The two tissue fractions were labeled with different fluorescent dyes and hybridized to an Agilent® CpG Island microarray to identify differentially methylated region [0384] [0368] [0269].
Regarding claims 8, 21 and 22, Melnikov discloses the first classification indicates that cancer exists for the organism, the method further comprising identifying a type of cancer associated with the organism. [0020] [0046]. Melnikov further discloses using thresholds for methylated fragments defining methylated and unmethylated calls for diagnosis of cancer [0090].
Melnikov discloses methods of identifying methylation patterns in genes associated with specific cancers (abstract). Melnikov further discloses a) reacting isolated genomic DNA from the subject and a methylation-sensitive restriction enzyme; wherein the genomic DNA comprises a plurality of promoters from different genes, and the enzyme cleaves unmethylated CpG sequences in the promoters and does not cleave methylated CpG sequences in the promoters; (b) contacting the genomic DNA thus reacted and a plurality of pairs of specific primers in an amplification mixture, the pairs of specific primers being configured to hybridize to the genomic DNA and to amplify a plurality of different promoters through a region comprising an uncleaved CpG sequence; (c) reacting the amplification mixture; (d) detecting one or more amplified promoters in the reacted amplification mixture or the absence thereof, thereby diagnosing cancer in the subject selected from the group consisting of ovarian cancer, lung cancer, prostate cancer, pancreatic cancer, and colon cancer (claim 1); a cell-free assay is provided in which a cancer marker gene, protein or biologically active portion thereof is contacted with a test compound and the ability of the test compound to bind to the cancer marker gene, protein or biologically active portion thereof is evaluated [0116]. Melnikov further discloses distinguishing normal from cancer status and cancer classification [0262-0268];
Regarding claim 27, Tynan discloses that the methylation-aware assay further comprises contacting the cell-free DNA molecules with a protein that binds methylated DNA [0211].
Regarding claims 31-35 and 37, Melnikov discloses comparing the first methylation level to a corresponding value determined from other organisms [0264-0267]; radiological and/or imaging investigation [0333-0334] [0004]; determining levels of one or more protein markers, wherein the one or more protein markers is selected from the group consisting of prostate specific antigen, carcinoembryonic antigen, alpha fetoprotein, CA125 and CA19-9 [0291-0293].
It would have been prima facie obvious to one of ordinary skill in the art, before the effective filing date of the claimed invention, to have modified the method of reference U.S. Patent No. 12,518,854 to have used methylation profiling method of Korshunova, Tynan, and Melnikov to classify abnormalities such as cancer. There would be a reasonable expectation of success in combining these methods because they all use methylation profiling to identify one or more abnormalities.
Claims 1-2, 4-7, 9-10, 12, 21-22, 25-26, and 28-31 are rejected on the ground of nonstatutory double patenting as being unpatentable over claims 1, 6-7, 9, 12-13, 16, 18, 29, and 31 of U.S. Patent application No. 16/930,231 in view of Korshunova et al. (2008: Massively parallel bisulphite pyrosequencing reveals the molecular complexity of breast cancer-associated cytosine-methylation patterns obtained from tissue and serum DNA, 18:19-29), in view of Tynan et al. (US20140093873A1), and further in view of Melnikov et al. (US20080261217A1).. Although the claims at issue are not identical, they are not patentably distinct from each other.
Regarding instant claims 1, 2, 21, 22, 29, and 30, reference claims 1 recites the limitations of said claims with extra limitations of performing a methylation-aware assay comprising enrichment of cell-free DNA molecules originating from specific genomic regions, wherein the specific genomic regions comprise genomic regions with a methylation differential between cancer and non-cancer for a tissue, wherein the enrichment comprises: (i) contacting the plurality of cell-free DNA molecules with hybridization probes to enrich for cell-free DNA molecules originating from the specific genomic regions, and determining sequences of the enriched cell-free DNA molecules; or (ii) performing methylation-specific PCR amplification of cell-free DNA molecules originating from the specific genomic regions.
Regarding instant claims 4 and 5, reference claims 6 and 7 recite treating the cell-free DNA molecules with the sodium bisulfite is part of Tet-assisted bisulfite conversion or oxidative bisulfite sequencing for a detection of 5-hydroxymethylcytosine.
Regarding instant claim 6, reference claim 12 recites determining a first classification of a level of cancer.
Regarding instant claim 7, reference claim 14 recites comparing the first methylation level to a cutoff value; and determining the first classification of cancer.
Regarding instant claim 9, reference claim 15 recites the cutoff value is a specified distance from a reference methylation level.
Regarding instant claim 10, reference claim 16 recites the specified distance is a specified number of standard deviations from the reference.
Regarding instant claim 12, reference claim 18 recites the specified distance.
Regarding instant claim 25, reference claim 9 recites the plurality of CpG sites are on a plurality of chromosomes.
Regarding instant claim 26, reference claim 29 recites the disjointed regions.
Regarding instant claim 28, reference claim 31 recites non-transitory computer readable medium comprising a plurality of instructions that, when executed, control a computer system to perform the method of claim 1.
Regarding instant claim 31, reference claim 13 recites comparing and identifying type of cancer.
Korshunova discloses that the locus-specific tag-bearing PCR products were enriched using coded primers, and amplified from bisulphate-modified DNA, purified, quantified individually, mixed in equimolar amounts, and sequenced using the 454-pyrosequencing platform (pg. 20, col. 2, last para.).
Tynan discloses non-invasive methylation-based enrichment of circulating DNA methods that utilize genomic regions that are differentially methylated between a mother and her fetus to separate, isolate or enrich fetal nucleic acid from a maternal sample non-invasively.
Tynan further discloses treating sample with a reagent that differentially modifies methylated and unmethylated DNA. For example, the reagent may comprise bisulfite; or the reagent may comprise one or more enzymes that preferentially cleave methylated DNA; or the reagent may comprise one or more enzymes that preferentially cleave unmethylated DNA (for example, methylation-aware assay) [0021].
Tynan discloses enriching a sample nucleic acid for a plurality of polymorphic nucleic acid targets (claim 1), where enriching comprises methylation sensitive capture, for example using, MBD2-Fc fragment; bisulfite conversion methods, for example, MSP (methylation-sensitive PCR), COBRA, methylation-sensitive single nucleotide primer extension (Ms-SNuPE) or Sequenom MassCLEAVE™ technology; and the use of methylation sensitive restriction enzymes [0031]. Tynan further discloses MSP (methylation-specific PCR) allows for assessing the methylation status and furter discloses that MSP requires only small quantities of DNA, is sensitive to 0.1% methylated alleles of a given CpG island locus, and can be performed on DNA extracted from paraffin-embedded samples. Typical reagents (e.g., as might be found in a typical MSP-based kit) for MSP analysis may include, but are not limited to: methylated and unmethylated PCR primers for specific gene (or methylation-altered DNA sequence or CpG island), optimized PCR buffers and deoxynucleotides, and specific probes [0225-0226] [0262-0282]. Tynan further discloses Nimblegen sequence capture system (Roche NimbleGen, Madison, Wis.); Illumina BEADARRAY platform (Illumina, San Diego, Calif.) (for example, targeted hybridization probe); Affymetrix GENECHIP platform (Affymetrix, Santa Clara, Calif.); Agilent SureSelect Target Enrichment System (Agilent Technologies, Santa Clara, Calif.) (for example, targeted hybridization probe) [0278].
Further regarding claim 29, Korshunova discloses reating the plurality of cell-free DNA molecules with sodium bisulfite (pg.22, col.1 last para.); Tynan discloses preparing the cfDNA comprises a hybridization process, a capture process, or an amplification process [0011]; Hyper- and hypomethylated nucleic acid sequences of the technology herein are identified [0014-0016]; see also quantitative methylation assay to determine methylation levels [0218].
Further regarding claim 30, Tynan discloses that MBD-FC was used to capture the methylated fraction of each DNA sample. See FIGS. 1-3. The two tissue fractions were labeled with different fluorescent dyes and hybridized to an Agilent® CpG Island microarray to identify differentially methylated region [0384] [0368] [0269].
Regarding claims 8, 21 and 22, Melnikov discloses the first classification indicates that cancer exists for the organism, the method further comprising identifying a type of cancer associated with the organism. [0020] [0046]. Melnikov further discloses using thresholds for methylated fragments defining methylated and unmethylated calls for diagnosis of cancer [0090].
Melnikov discloses methods of identifying methylation patterns in genes associated with specific cancers (abstract).
It would have been prima facie obvious to one of ordinary skill in the art, before the effective filing date of the claimed invention, to have modified the method of reference U.S. Patent application No. 16/930,231 to have used methylation profiling method of Korshunova, Tynan, and Melnikov to classify abnormalities such as cancer. There would be a reasonable expectation of success in combining these methods because they all use methylation profiling to identify one or more abnormalities.
Claims 1-2, 4-8, 12, 21-25, 28, and 31 are rejected on the ground of nonstatutory double patenting as being unpatentable over claims 1-2, 6-10, 14, 23-27, and 30 of U.S. Patent No. 10,706,957 in view of Korshunova et al. (2008: Massively parallel bisulphite pyrosequencing reveals the molecular complexity of breast cancer-associated cytosine-methylation patterns obtained from tissue and serum DNA, 18:19-29), in view of Tynan et al. (US20140093873A1), and further in view of Melnikov et al. (US20080261217A1).. Although the claims at issue are not identical, they are not patentably distinct from each other.
Regarding instant claims 1, 2, 21, 22, 29, and 30, reference claims 1 recites the limitations of said claims with extra limitations of performing a methylation-aware assay comprising enrichment of cell-free DNA molecules originating from specific genomic regions, wherein the specific genomic regions comprise genomic regions with a methylation differential between cancer and non-cancer for a tissue, wherein the enrichment comprises: (i) contacting the plurality of cell-free DNA molecules with hybridization probes to enrich for cell-free DNA molecules originating from the specific genomic regions, and determining sequences of the enriched cell-free DNA molecules; or (ii) performing methylation-specific PCR amplification of cell-free DNA molecules originating from the specific genomic regions.
Regarding instant claim 2, reference claims 2 recites sequencing of at least 60,000 cell-free DNA.
Regarding instant claims 4 and 5, reference claims 6 and 7 recite treating the cell-free DNA molecules with sodium bisulfite is part of Tet-assisted bisulfite conversion or oxidative bisulfite sequencing for a detection of 5-hydroxymethylcytosine.
Regarding instant claims 6 and 7, reference claims 8 and 9 recite determining the first classification.
Regarding instant claim 8, reference claims 10 recites determining the type of cancer.
Regarding instant claim 12, reference claims 14 recites comparing the first methylation level to the first cutoff value.
Regarding instant claims 21 and 22, reference claims 23 and 24 recite the plurality of sites includes CpG site.
Regarding instant claims 23 and 24, reference claims 25 and 26 recite determining a fractional concentration of tumor DNA.
Regarding instant claim 25, reference claims 27 recites the plurality of sites are on a plurality of chromosomes.
Regarding instant claim 28, reference claims 30 recite a computer system.
Regarding instant claim 31, reference claims 10 recite the type of cancer.
Korshunova discloses that the locus-specific tag-bearing PCR products were enriched using coded primers, and amplified from bisulphate-modified DNA, purified, quantified individually, mixed in equimolar amounts, and sequenced using the 454-pyrosequencing platform (pg. 20, col. 2, last para.).
Tynan discloses non-invasive methylation-based enrichment of circulating DNA methods that utilize genomic regions that are differentially methylated between a mother and her fetus to separate, isolate or enrich fetal nucleic acid from a maternal sample non-invasively.
Tynan further discloses treating sample with a reagent that differentially modifies methylated and unmethylated DNA. For example, the reagent may comprise bisulfite; or the reagent may comprise one or more enzymes that preferentially cleave methylated DNA; or the reagent may comprise one or more enzymes that preferentially cleave unmethylated DNA (for example, methylation-aware assay) [0021].
Tynan discloses enriching a sample nucleic acid for a plurality of polymorphic nucleic acid targets (claim 1), where enriching comprises methylation sensitive capture, for example using, MBD2-Fc fragment; bisulfite conversion methods, for example, MSP (methylation-sensitive PCR), COBRA, methylation-sensitive single nucleotide primer extension (Ms-SNuPE) or Sequenom MassCLEAVE™ technology; and the use of methylation sensitive restriction enzymes [0031]. Tynan further discloses MSP (methylation-specific PCR) allows for assessing the methylation status and furter discloses that MSP requires only small quantities of DNA, is sensitive to 0.1% methylated alleles of a given CpG island locus, and can be performed on DNA extracted from paraffin-embedded samples. Typical reagents (e.g., as might be found in a typical MSP-based kit) for MSP analysis may include, but are not limited to: methylated and unmethylated PCR primers for specific gene (or methylation-altered DNA sequence or CpG island), optimized PCR buffers and deoxynucleotides, and specific probes [0225-0226] [0262-0282]. Tynan further discloses Nimblegen sequence capture system (Roche NimbleGen, Madison, Wis.); Illumina BEADARRAY platform (Illumina, San Diego, Calif.) (for example, targeted hybridization probe); Affymetrix GENECHIP platform (Affymetrix, Santa Clara, Calif.); Agilent SureSelect Target Enrichment System (Agilent Technologies, Santa Clara, Calif.) (for example, targeted hybridization probe) [0278].
Further regarding claim 29, Korshunova discloses reating the plurality of cell-free DNA molecules with sodium bisulfite (pg.22, col.1 last para.); Tynan discloses preparing the cfDNA comprises a hybridization process, a capture process, or an amplification process [0011]; Hyper- and hypomethylated nucleic acid sequences of the technology herein are identified [0014-0016]; see also quantitative methylation assay to determine methylation levels [0218].
Further regarding claim 30, Tynan discloses that MBD-FC was used to capture the methylated fraction of each DNA sample. See FIGS. 1-3. The two tissue fractions were labeled with different fluorescent dyes and hybridized to an Agilent® CpG Island microarray to identify differentially methylated region [0384] [0368] [0269].
Regarding claims 8, 21 and 22, Melnikov discloses the first classification indicates that cancer exists for the organism, the method further comprising identifying a type of cancer associated with the organism. [0020] [0046]. Melnikov further discloses using thresholds for methylated fragments defining methylated and unmethylated calls for diagnosis of cancer [0090].
Melnikov discloses methods of identifying methylation patterns in genes associated with specific cancers (abstract).
It would have been prima facie obvious to one of ordinary skill in the art, before the effective filing date of the claimed invention, to have modified the method of reference U.S. Patent No. 107,06,957 to have used methylation profiling method of Korshunova, Tynan, and Melnikov to classify abnormalities such as cancer. There would be a reasonable expectation of success in combining these methods because they all use methylation profiling to identify one or more abnormalities.
Claims 1, 2, 4-10, 12, 21-24, and 29-31 are rejected on the ground of nonstatutory double patenting as being unpatentable over claims 1, 3-4, 11, 13, 38-39, 43-44, 54, 56, and 81 of U.S. Patent No. 10,392,666 in view of Korshunova et al. (2008: Massively parallel bisulphite pyrosequencing reveals the molecular complexity of breast cancer-associated cytosine-methylation patterns obtained from tissue and serum DNA, 18:19-29), in view of Tynan et al. (US20140093873A1), and further in view of Melnikov et al. (US20080261217A1).. Although the claims at issue are not identical, they are not patentably distinct from each other.
Regarding instant claims 1, 29, and 30, reference claims 1 and 3 recite the limitations of said claims with extra limitations of enrichment of cell-free DNA molecules originating from specific genomic regions, wherein the specific genomic regions comprise genomic regions with a methylation differential between cancer and non-cancer for a tissue, wherein the enrichment comprises: (i) contacting the plurality of cell-free DNA molecules with hybridization probes to enrich for cell-free DNA molecules originating from the specific genomic regions, and determining sequences of the enriched cell-free DNA molecules; or (ii) performing methylation-specific PCR amplification of cell-free DNA molecules originating from the specific genomic regions; treating the cell-free DNA molecules with sodium bisulfite;
Regarding instant claim 2, reference claim 81 recite the massively parallel sequencing generates at least 60,000 sequence reads.
Regarding instant claims 4 and 5, reference claim 3 and 4 recite treating the cell-free DNA molecules with sodium bisulfite.
Regarding instant claims 6 and 7, reference claim 1 recite a first classification.
Regarding instant claim 8, reference claim 11 recite a type of cancer.
Regarding instant claim 9, reference claim 38 recite the first cutoff value is a specified distance.
Regarding instant claim 10, reference claim 39 recite the specified distance is a specified number of standard deviations.
Regarding instant claim 12, reference claim 13 recite determining a difference, and comparing the difference to a threshold.
Regarding instant claim 21, reference claim 54 recite the plurality of sites includes CpG sites.
Regarding instant claim 22, reference claim 56 recite calculating a cumulative score from the respective methylation densities; determine the first classification.
Regarding instant claim 23, reference claim 43 recite lagging the biological sample.
Regarding instant claim 24, reference claim 44 recite the minimum value is determined based on an expected difference.
Regarding instant claim 31, reference claim 11 recite identifying a type of cancer.
Korshunova discloses that the locus-specific tag-bearing PCR products were enriched using coded primers, and amplified from bisulphate-modified DNA, purified, quantified individually, mixed in equimolar amounts, and sequenced using the 454-pyrosequencing platform (pg. 20, col. 2, last para.).
Tynan discloses non-invasive methylation-based enrichment of circulating DNA methods that utilize genomic regions that are differentially methylated between a mother and her fetus to separate, isolate or enrich fetal nucleic acid from a maternal sample non-invasively.
Tynan further discloses treating sample with a reagent that differentially modifies methylated and unmethylated DNA. For example, the reagent may comprise bisulfite; or the reagent may comprise one or more enzymes that preferentially cleave methylated DNA; or the reagent may comprise one or more enzymes that preferentially cleave unmethylated DNA (for example, methylation-aware assay) [0021].
Tynan discloses enriching a sample nucleic acid for a plurality of polymorphic nucleic acid targets (claim 1), where enriching comprises methylation sensitive capture, for example using, MBD2-Fc fragment; bisulfite conversion methods, for example, MSP (methylation-sensitive PCR), COBRA, methylation-sensitive single nucleotide primer extension (Ms-SNuPE) or Sequenom MassCLEAVE™ technology; and the use of methylation sensitive restriction enzymes [0031]. Tynan further discloses MSP (methylation-specific PCR) allows for assessing the methylation status and furter discloses that MSP requires only small quantities of DNA, is sensitive to 0.1% methylated alleles of a given CpG island locus, and can be performed on DNA extracted from paraffin-embedded samples. Typical reagents (e.g., as might be found in a typical MSP-based kit) for MSP analysis may include, but are not limited to: methylated and unmethylated PCR primers for specific gene (or methylation-altered DNA sequence or CpG island), optimized PCR buffers and deoxynucleotides, and specific probes [0225-0226] [0262-0282]. Tynan further discloses Nimblegen sequence capture system (Roche NimbleGen, Madison, Wis.); Illumina BEADARRAY platform (Illumina, San Diego, Calif.) (for example, targeted hybridization probe); Affymetrix GENECHIP platform (Affymetrix, Santa Clara, Calif.); Agilent SureSelect Target Enrichment System (Agilent Technologies, Santa Clara, Calif.) (for example, targeted hybridization probe) [0278].
Further regarding claim 29, Korshunova discloses reating the plurality of cell-free DNA molecules with sodium bisulfite (pg.22, col.1 last para.); Tynan discloses preparing the cfDNA comprises a hybridization process, a capture process, or an amplification process [0011]; Hyper- and hypomethylated nucleic acid sequences of the technology herein are identified [0014-0016]; see also quantitative methylation assay to determine methylation levels [0218].
Further regarding claim 30, Tynan discloses that MBD-FC was used to capture the methylated fraction of each DNA sample. See FIGS. 1-3. The two tissue fractions were labeled with different fluorescent dyes and hybridized to an Agilent® CpG Island microarray to identify differentially methylated region [0384] [0368] [0269].
Regarding claims 8, 21 and 22, Melnikov discloses the first classification indicates that cancer exists for the organism, the method further comprising identifying a type of cancer associated with the organism. [0020] [0046]. Melnikov further discloses using thresholds for methylated fragments defining methylated and unmethylated calls for diagnosis of cancer [0090].
Melnikov discloses methods of identifying methylation patterns in genes associated with specific cancers (abstract).
It would have been prima facie obvious to one of ordinary skill in the art, before the effective filing date of the claimed invention, to have modified the method of reference U.S. Patent No. 10,392,666 to have used methylation profiling method of Korshunova, Tynan, and Melnikov to classify abnormalities such as cancer. There would be a reasonable expectation of success in combining these methods because they all use methylation profiling to identify one or more abnormalities.
Claims 11, 4, 6-7, 21, and 29-30 are rejected on the ground of nonstatutory double patenting as being unpatentable over claims 1-2, 8, and 16-17 of U.S. Patent No. 9,732,390 in view of Korshunova et al. (2008: Massively parallel bisulphite pyrosequencing reveals the molecular complexity of breast cancer-associated cytosine-methylation patterns obtained from tissue and serum DNA, 18:19-29), in view of Tynan et al. (US20140093873A1), and further in view of Melnikov et al. (US20080261217A1).. Although the claims at issue are not identical, they are not patentably distinct from each other.
Regarding instant claims 1, 29, and 30, reference claims 1 and 17 recite the limitations of said claims with extra limitations of enrichment of cell-free DNA molecules originating from specific genomic regions, wherein the specific genomic regions comprise genomic regions with a methylation differential between cancer and non-cancer for a tissue, wherein the enrichment comprises: (i) contacting the plurality of cell-free DNA molecules with hybridization probes to enrich for cell-free DNA molecules originating from the specific genomic regions, and determining sequences of the enriched cell-free DNA molecules; or (ii) performing methylation-specific PCR amplification of cell-free DNA molecules originating from the specific genomic regions; treating the cell-free DNA molecules with sodium bisulfite;
Regarding instant claim 4, reference claim 17 recites treating the cell-free DNA with sodium bisulfite.
Regarding instant claims 6 and 7, reference claim 2 recites determine a classification of a level of cancer.
Regarding instant claim 21, reference claims 8 and 16 recite the one or more sites are CpG sites.
Korshunova discloses that the locus-specific tag-bearing PCR products were enriched using coded primers, and amplified from bisulphate-modified DNA, purified, quantified individually, mixed in equimolar amounts, and sequenced using the 454-pyrosequencing platform (pg. 20, col. 2, last para.).
Tynan discloses non-invasive methylation-based enrichment of circulating DNA methods that utilize genomic regions that are differentially methylated between a mother and her fetus to separate, isolate or enrich fetal nucleic acid from a maternal sample non-invasively.
Tynan further discloses treating sample with a reagent that differentially modifies methylated and unmethylated DNA. For example, the reagent may comprise bisulfite; or the reagent may comprise one or more enzymes that preferentially cleave methylated DNA; or the reagent may comprise one or more enzymes that preferentially cleave unmethylated DNA (for example, methylation-aware assay) [0021].
Tynan discloses enriching a sample nucleic acid for a plurality of polymorphic nucleic acid targets (claim 1), where enriching comprises methylation sensitive capture, for example using, MBD2-Fc fragment; bisulfite conversion methods, for example, MSP (methylation-sensitive PCR), COBRA, methylation-sensitive single nucleotide primer extension (Ms-SNuPE) or Sequenom MassCLEAVE™ technology; and the use of methylation sensitive restriction enzymes [0031]. Tynan further discloses MSP (methylation-specific PCR) allows for assessing the methylation status and furter discloses that MSP requires only small quantities of DNA, is sensitive to 0.1% methylated alleles of a given CpG island locus, and can be performed on DNA extracted from paraffin-embedded samples. Typical reagents (e.g., as might be found in a typical MSP-based kit) for MSP analysis may include, but are not limited to: methylated and unmethylated PCR primers for specific gene (or methylation-altered DNA sequence or CpG island), optimized PCR buffers and deoxynucleotides, and specific probes [0225-0226] [0262-0282]. Tynan further discloses Nimblegen sequence capture system (Roche NimbleGen, Madison, Wis.); Illumina BEADARRAY platform (Illumina, San Diego, Calif.) (for example, targeted hybridization probe); Affymetrix GENECHIP platform (Affymetrix, Santa Clara, Calif.); Agilent SureSelect Target Enrichment System (Agilent Technologies, Santa Clara, Calif.) (for example, targeted hybridization probe) [0278].
Further regarding claim 29, Korshunova discloses reating the plurality of cell-free DNA molecules with sodium bisulfite (pg.22, col.1 last para.); Tynan discloses preparing the cfDNA comprises a hybridization process, a capture process, or an amplification process [0011]; Hyper- and hypomethylated nucleic acid sequences of the technology herein are identified [0014-0016]; see also quantitative methylation assay to determine methylation levels [0218].
Further regarding claim 30, Tynan discloses that MBD-FC was used to capture the methylated fraction of each DNA sample. See FIGS. 1-3. The two tissue fractions were labeled with different fluorescent dyes and hybridized to an Agilent® CpG Island microarray to identify differentially methylated region [0384] [0368] [0269].
Regarding claims 8, 21 and 22, Melnikov discloses the first classification indicates that cancer exists for the organism, the method further comprising identifying a type of cancer associated with the organism. [0020] [0046]. Melnikov further discloses using thresholds for methylated fragments defining methylated and unmethylated calls for diagnosis of cancer [0090].
Melnikov discloses methods of identifying methylation patterns in genes associated with specific cancers (abstract).
It would have been prima facie obvious to one of ordinary skill in the art, before the effective filing date of the claimed invention, to have modified the method of reference U.S. Patent No. 9,732,390 to have used methylation profiling method of Korshunova, Tynan, and Melnikov to classify abnormalities such as cancer. There would be a reasonable expectation of success in combining these methods because they all use methylation profiling to identify one or more abnormalities.
Conclusion
No claims are allowed.
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/G.S./Examiner, Art Unit 1686
/LARRY D RIGGS II/Supervisory Patent Examiner, Art Unit 1686