DETAILED ACTION
The amendment filed on 01/13/2026 has been entered.
Claims 1, 3-11 and 13-15 were amended in the claim set filed on 01/13/2026.
Applicant’s election without traverse of Group I (Claims 1-16) and species (SLC11A1, NLRP12, PRTN3, HMBS, LILRB3, ACSL1, GP9, MX2, RASGRP4, ATG16L2) in the reply filed on 05/15/25 is acknowledged. Because applicant did not distinctly and specifically point out the supposed errors in the restriction requirement, the election has been treated as an election without traverse (MPEP § 818.01(a)).
Claim 16 is withdrawn from further consideration pursuant to 37 CFR 1.142(b) as being drawn to a nonelected species, there being no allowable generic or linking claim. Election was made without traverse in the reply filed on 07/09/2025.
Claims Status
Claims 2 and 12 are canceled
Claims 1, 3-11 and 13-20 are pending.
Claims 16-20 are withdrawn.
Claims 1, 3-11 and 13-15 are currently under examination
Response to the Arguments
Objections to the Specification in the previously mailed non-final have been withdrawn in light of applicants submission of abstract on a separate sheet.
Applicant’s arguments regarding previous rejections of claims 1, 3-11 and 12-15 under 35 U.S.C. § 112 have been fully considered and are persuasive in part. Most of the 35 U.S.C. 112 rejections documented in the previously mailed non-final have been withdrawn in light of applicants claim amendments and arguments on Pg. 7-12. The remaining revised rejection under 35 U.S.C. § 112 (b) considered not persuasive is documented below on Pg. 4.
Applicant’s arguments regarding previous rejections of claims 1, 3-11 and 12-15 under 35 U.S.C. 101 have been fully considered and are not persuasive. The 35 U.S.C. 101 rejections documented in the previously mailed non-final have been maintained and revised in light of applicants claim amendments and arguments on Pg. 12-13. As necessitated by amendment, revised rejections for claims 1, 3-11 and 13-15 are made, as documented below, under the 35 U.S.C. 101 rejections in this office action on Pg. 4-11.
Applicant’s arguments regarding previous rejections of claims 1, 3-11 and 12-15under 35 U.S.C. 102 and 35 U.S.C. 103 have been fully considered and are persuasive in light of applicants claim amendments and arguments. Previous rejections of claims 1, 3-11 and 12-15 under 35 U.S.C. 102 and 35 U.S.C. 103 have been withdrawn. However, as necessitated by amendments, new grounds of rejection for claims 1, 3-11 and 13-15 are made, as documented below, under the 35 U.S.C. 103 rejections in this office action on Pg. 12-23.
The rejections for claims 1, 3-11 and 12-15 are documented below in this Final Office Action are necessitated by claim amendments filed on 01/13/2026.
Priority
This application is a 371 U.S. National Stage of International Application No.
PCT/SG2020/050766, filed December 18, 2020, and claims priority to Singapore Application No. 10201912600T, filed December 19, 2019.
Acknowledgment is made of applicant' s claim for foreign priority under 35 U.S.C. 119 (a)-(d). The certified copy of SG10201912600T and English translation has been submitted of the record on June 10, 2022. Accordingly, the priority date of claim set filed on June 10, 2022 is determined to be Dec. 19, 2019.
Claim Objections
Claim 1 is objected to because of the following informalities: “;and” (ln 6) should be amended to “;” . Appropriate correction is required.
Claim Rejections - 35 USC § 112 (b)
The following is a quotation of 35 U.S.C. 112(b):
(b) CONCLUSION.—The specification shall conclude with one or more claims particularly pointing out and distinctly claiming the subject matter which the inventor or a joint inventor regards as the invention.
The following is a quotation of 35 U.S.C. 112 (pre-AIA ), second paragraph:
The specification shall conclude with one or more claims particularly pointing out and distinctly claiming the subject matter which the applicant regards as his invention.
Claims 1, 3-11 and 13-15 remain/are rejected under 35 U.S.C. 112(b) or 35 U.S.C. 112 (pre-AIA ), second paragraph, as being incomplete for omitting essential steps, such omission amounting to a gap between the steps. See MPEP § 2172.01. The omitted steps are: With regard to claims 1-3, steps (1) cfDNA fragmentation and (2) isolation of cfDNA fragments. 3-15 depend on claim 1.
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, 3-11 and 13-15 remain/are rejected under 35 U.S.C. 101 because the claimed invention is directed towards an abstract ideas of quantifying the ctDNA burden based on said level of cfDNA using a model, comparing the ctDNA burden and identifying the diseased /tumor-bearing subject as having disease progression, a natural correlation of cfDNA levels to disease progression and routine and conventional steps of sequencing cfDNA fragments and quantifying ctDNA burden, without significantly more. The claims recite abstract ideas, a natural phenomenon and routine and conventional methods. This judicial exception is not integrated into a practical application because no additional elements integrate the judicial exceptions into a practical application. The claims do not include additional elements that are sufficient to amount to significantly more than the judicial exception because no additional elements are considered significantly more than the judicial exceptions.
Claim analysis
The instant claim 1 is directed towards: “A method of quantifying circulating tumor DNA (ctDNA) burden in a diseased /tumor-bearing subject, the method comprising: determining in a blood sample obtained from a healthy subject and the diseased / tumor-bearing subject, a level of cell-free DNA (cfDNA) that maps to six or more nucleosome-depleted regions (NDR) comprising; sequencing cfDNA fragments in the blood sample to obtain sequencing reads; and determining the number of sequencing reads that align with the six or more NDRs to obtain said level of cfDNA that maps to the six or more NDRs; and quantifying the ctDNA burden based on said level of cfDNA using a model, wherein said NDRs (i) comprises the six or more NDRs of a-six or more genes which transcripts are differentially expressed between a healthy blood sample and a tumor blood sample and/or (ii) are degraded to different extents between cfDNA in the healthy blood sample and cfDNA in the blood-sample of the diseased /tumor-bearing subject, wherein the six or more NDRs comprises the six or more NDRs of genes selected from the group consisting of: SLC11A1, NLRP12, PRTN3, HMBS, LILRB3, ACSL1,GP9, MX2, RASGRP4, ATG16L2 and combinations thereof.
The “quantifying the ctDNA burden based on said level of cfDNA using a model” is a process of organizing information through mathematical correlations which is directed to an abstract idea.
Claim 1 implicitly correlates the differentially expressed transcript of a NDR to a tumor sample.
The sequencing cfDNA fragments and quantifying ctDNA burden are considered to be active steps requiring the analysis of a sample. The active step is routine and conventional as demonstrated by the 35 USC § 103 rejections stated below.
Dependent claims of claim 1 set forth further limitations about cfDNA levels, cfDNA preparation, NDR(s), quantitated ctDNA burden, transcript, disease progression, and treatment regimen.
The instant claim 13 is directed towards: The method according to claim 1, wherein the method further comprises determining disease progression in the diseased /tumor-bearing subject comprising: determining in a subsequent blood sample obtained from the diseased / tumor- bearing subject, a level of cfDNA that maps to six or more NDRs; quantifying the ctDNA burden based on said level of cfDNA; comparing the ctDNA burden quantified from said subsequent blood sample with the ctDNA burden quantified from said blood sample; and identifying the diseased /tumor-bearing subject as having disease progression if the ctDNA burden quantified from said subsequent blood sample is higher than the ctDNA burden quantified from said blood sample and if the ctDNA burden quantified from said subsequent blood sample is not higher than the ctDNA burden quantified from said blood sample, the disease is identified to be improving/ablating in the diseased /tumor-bearing subject; wherein disease progression in the diseased /tumor-bearing subject indicates resistance to a treatment regimen received by the diseased /tumor-bearing subject.
The “comparing the ctDNA burden quantified from said subsequent blood sample with the ctDNA burden quantified from said blood sample” is an abstract idea.
The “identifying the diseased /tumor-bearing subject as having disease progression if the ctDNA burden quantified from said subsequent blood sample is higher than the ctDNA burden quantified from said blood sample and if the ctDNA burden quantified from said subsequent blood sample is not higher than the ctDNA burden quantified from said blood sample, the disease is identified to be improving/ablating in the diseased /tumor-bearing subject; wherein disease progression in the diseased /tumor-bearing subject indicates resistance to a treatment regimen received by the diseased /tumor-bearing subject” is an abstract idea.
The correlation of cfDNA levels to disease progression is a natural phenomenon.
The quantifying ctDNA burden, without significantly more. is considered to be an active step requiring the analysis of a sample. The active step is routine and conventional as demonstrated by the 35 USC § 103 rejections stated below.
Dependent claim 14 is directed to: The method according to claim 13, the method further comprising changing the treatment regimen received by the diseased / tumor-bearing subject if the diseased / tumor-bearing subject is identified as having disease progression.
The treatment step is not a particular treatment step.
“changing the treatment regimen received by the diseased / tumor-bearing subject if the diseased / tumor-bearing subject is identified as having disease progression” is considered to be an active step requiring the treatment of a subject if disease progression is identified. The active step is routine and conventional as demonstrated by the 35 USC § 103 rejections stated below and
According to the 2019 Patent Eligibility Guidance an initial two step analysis is required for determining statutory eligibility.
Step 1. Is the claim directed to a process, machine, manufacture, or composition of matter? In the instant case, the Step 1 requirement is satisfied as the claims are directed towards a process.
Step 2A Prong one. Does the claim recite a law of nature, a natural phenomenon or an abstract idea? Yes, abstract ideas and natural phenomenon.
With regard to claim 1, the claim recites, “A method of quantifying circulating tumor DNA (ctDNA) burden in a diseased /tumor-bearing subject, the method comprising: determining in a blood sample obtained from a healthy subject and the diseased / tumor-bearing subject, a level of cell-free DNA (cfDNA) that maps to six or more nucleosome-depleted regions (NDR) comprising; sequencing cfDNA fragments in the blood sample to obtain sequencing reads; and determining the number of sequencing reads that align with the six or more NDRs to obtain said level of cfDNA that maps to the six or more NDRs; and quantifying the ctDNA burden based on said level of cfDNA using a model, wherein said NDRs (i) comprises the six or more NDRs of a-six or more genes which transcripts are differentially expressed between a healthy blood sample and a tumor blood sample and/or (ii) are degraded to different extents between cfDNA in the healthy blood sample and cfDNA in the blood-sample of the diseased /tumor-bearing subject, wherein the six or more NDRs comprises the six or more NDRs of genes selected from the group consisting of: SLC11A1, NLRP12, PRTN3, HMBS, LILRB3, ACSL1,GP9, MX2, RASGRP4, ATG16L2 and combinations thereof. The “quantifying the ctDNA burden based on said level of cfDNA using a model” is a process of organizing information through mathematical correlations which is directed to an abstract idea.
With regard to claim 13, the claim recites, “The method according to claim 1, wherein the method further comprises determining disease progression in the diseased /tumor-bearing subject comprising: determining in a subsequent blood sample obtained from the diseased / tumor- bearing subject, a level of cfDNA that maps to six or more NDRs; quantifying the ctDNA burden based on said level of cfDNA; comparing the ctDNA burden quantified from said subsequent blood sample with the ctDNA burden quantified from said blood sample; and identifying the diseased /tumor-bearing subject as having disease progression if the ctDNA burden quantified from said subsequent blood sample is higher than the ctDNA burden quantified from said blood sample and if the ctDNA burden quantified from said subsequent blood sample is not higher than the ctDNA burden quantified from said blood sample, the disease is identified to be improving/ablating in the diseased /tumor-bearing subject; wherein disease progression in the diseased /tumor-bearing subject indicates resistance to a treatment regimen received by the diseased /tumor-bearing subject.” The “comparing the ctDNA burden estimated from said subsequent blood sample with the ctDNA burden estimated from said blood sample” is an abstract idea. The correlation of cfDNA levels to disease progression is a natural phenomenon.
Step 2A prong two. Does the claim recite additional elements that integrate the judicial exception into a practical application? No, there are no additional steps that integrate the claims into a practical application.
Step 2B. Does the claim recite additional elements that are significantly more than the judicial exceptions? No, there are no additional elements that are significantly more than the judicial exceptions.
Regarding claim 1, the claim requires the routine and conventional active steps of sequencing cfDNA fragments and quantifying ctDNA burden similar to that of Shendure et al. (“Shendure”; Patent App. Pub. WO 2016015058 A2, Jan.28. 2016) in view of Diehn et al. (“Diehn”; Patent App. Pub. WO 2014151117 A1, Sept. 25, 2014) and Huang et al. (“Huang”; (2019). Bioinformatics Analysis for Circulating Cell-Free DNA in Cancer. Cancers, 11(6), 805, June 10, 2019).
Shendure discloses “methods of determining one or more tissues and/or cell-types contributing to cell-free DNA ("cfDNA") in a biological sample of a subject. In some embodiments, the present disclosure provides a method of identifying a disease or disorder in a subject as a function of one or more determined more tissues and/or cell-types contributing to cfDNA in a biological sample from the subject.” (Abstract).
Diehn discloses “Methods for creating a selector of mutated genomic regions and for using the selector set to analyze genetic alterations in a cell-free nucleic acid sample are provided. The methods can be used to measure tumor-derived nucleic acids in a blood sample from a subject and thus to monitor the progression of disease in the subject. The methods can also be used for cancer screening, cancer diagnosis, cancer prognosis, and cancer therapy designation.” (Abstract).
Huang discloses “Molecular analysis of cell-free DNA (cfDNA) that circulates in plasma and other body fluids represents a “liquid biopsy” approach for non-invasive cancer screening or monitoring. The rapid development of sequencing technologies has made cfDNA a promising source to study cancer development and progression. Specific genetic and epigenetic alterations have been found in plasma, serum, and urine cfDNA and could potentially be used as diagnostic or prognostic biomarkers in various cancer types. In this review, we will discuss the molecular characteristics of cancer cfDNA and major bioinformatics approaches involved in the analysis of cfDNA sequencing data for detecting genetic mutation, copy number alteration, methylation change, and nucleosome positioning variation. We highlight specific challenges in sensitivity to detect genetic aberrations and robustness of statistical analysis. Finally, we provide perspectives regarding the standard and continuing development of bioinformatics analysis to move this promising screening tool into clinical practice.” (Abstract).
Thus, the claim does not provide additional steps which are significantly more.
Dependent claims require additional limitations of cfDNA preparation, NDRs, quantitation and alignment of sequencing reads, transcript expression, quantification of ctDNA burden, identification of disease progression, and changing treatment regimen, which are all routine and conventional based on Shendure et al. (“Shendure”; Patent App. Pub. WO 2016015058 A2, Jan.28. 2016), Diehn et al. (“Diehn”; Patent App. Pub. WO 2014151117 A1, Sept. 25, 2014) and Huang et al. (“Huang”; (2019). Bioinformatics Analysis for Circulating Cell-Free DNA in Cancer. Cancers, 11(6), 805, June 10, 2019) and demonstrated by the 35 USC § 103 rejections stated below.
Response to Arguments
Applicant's arguments filed 01/13/2026 (Pg. 12-13) with respect to claims 1, 3-11 and 13-15 have been fully considered but they are not persuasive. To clarify some instances argued in the response filed 01/13/2026 see responses to each argument made by Applicant below:
Applicants’ argument: “amended claim 1 and its dependent claims recite a non-natural molecular panel, with specific active steps such as sequencing, fragmenting which can be integrated into a practical application of quantifying a ctDNA burden in a diseased/ tumor-bearing subject”
Response: Applicant’s arguments have been fully considered and found unpersuasive because applicants amendments do not overcome the lack of patentably matter under U.S.C. 35 101. The previously presented and amended claims recite abstract ideas requiring a model (mathematical relationship) and routine and conventional steps of determining in a blood sample obtained from a healthy and the diseased subject, a level of cell-free DNA (cfDNA) that maps to six or more nucleosome-depleted region (NDR) comprising sequencing and quantifying ctDNA burden. The judicial exceptions are not integrated into a practical application because the claim limitations do not appear to improve the current technology or technical field beyond well-understood, routine, conventional activity. The claims do not include additional elements that are sufficient to amount to significantly more than the judicial exception because the claims provide no specific limitations that provide 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.
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, 3-11 and 13-15 are rejected under 35 U.S.C. 103 as being unpatentable over Shendure et al. (“Shendure”; Patent App. Pub. WO 2016015058 A2, Jan.28. 2016) in view of Diehn et al. (“Diehn”; Patent App. Pub. WO 2014151117 A1, Sept. 25, 2014) and Huang et al. (“Huang”; (2019). Bioinformatics Analysis for Circulating Cell-Free DNA in Cancer. Cancers, 11(6), 805, June 10, 2019).
Interpretation: Regarding claim 1, nucleosome depleted region (NDR) is interpreted as any genomic region that has relatively low nucleosome occupancy level as stated on Pg. 9 of the instant application specification.
Shendure discloses “methods of determining one or more tissues and/or cell-types contributing to cell-free DNA ("cfDNA") in a biological sample of a subject. In some embodiments, the present disclosure provides a method of identifying a disease or disorder in a subject as a function of one or more determined more tissues and/or cell-types contributing to cfDNA in a biological sample from the subject.” (Abstract).
Regarding claim 1, Shendure teaches a method wherein “a method of identifying a disease or disorder in a subject, the method comprising isolating cell-free DNA (cfDNA) from a biological sample from the subject, the isolated cfDNA comprising a plurality of cfDNA fragments; determining a sequence associated with at least a portion of the plurality of cfDNA fragments; determining a genomic location within a reference genome for at least some cfDNA fragment endpoints of the plurality of cfDNA fragments as a function of the cfDNA fragment sequences; determining at least some of the tissues and/or cell types giving rise to the cfDNA as a function of the genomic locations of at least some of the cfDNA fragment endpoints; and identifying the disease or disorder as a function of the determined tissues and/or cell types giving rise to the cfDNA.” (Para. 8) and “the method comprising: (i) generating a nucleosome map by obtaining a biological sample from the subject, isolating the cfDNA from the biological sample, and measuring distributions (a), (b) and/or (c) by library construction and massively parallel sequencing of cfDNA; (ii) generating a reference set of nucleosome maps by obtaining a biological sample from control subjects or subjects with known disease, isolating the cfDNA from the biological sample, measuring distributions (a), (b) and/or (c) by library construction and massively parallel sequencing of cfDNA; and (iii) determining tissues and/or cell types giving rise to the cfDNA from the biological sample by comparing the nucleosome map derived from the cfDNA from the biological sample to the reference set of nucleosome maps; wherein (a), (b) and (c) are: (a) the distribution of likelihoods any specific base-pair in a human genome will appear at a terminus of a cfDNA fragment; (b) the distribution of likelihoods that any pair of base-pairs of a human genome will appear as a pair of termini of a cfDNA fragment; and (c) the distribution of likelihoods that any specific base-pair in a human genome will appear in a cfDNA fragment as a consequence of differential nucleosome occupancy.” (Para. 9).
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Regarding claim 1, Shendure teaches a method comprising “the biological sample comprises, consists essentially of, or consists of whole blood, peripheral blood plasma” (Para. 85) Shendure teaches a method comprising “Specifically, it was expected that cfDNA fragment endpoints should cluster adjacent to nucleosome boundaries, while also being depleted on the nucleosome itself. To quantify this, the WPS was developed, which represents the number of DNA fragments completely spanning a 120 bp window centered at a given genomic coordinate, minus the number of fragments with an endpoint within that same window (FIG. 25)”. (Para. 183; Fig. 25 see below). Shendure teaches a method comprising “Nucleosomes are known to be well-positioned in relation to landmarks of gene regulation, for example transcriptional start sites and exon-intron boundaries.”(Para. 188). Shendure teaches a method comprising “one or more genes” (Para. 88; Para. 109). “cfDNA fragment endpoints should cluster adjacent to nucleosome boundaries, while also being depleted on the nucleosome itself”(Para. 183) and “exon-intron boundaries” (Para. 183) read on comprising a nucleosome depleted region.
Regarding claim 1, Shendure teaches a method wherein “one or more genes” (Para. 88; Para. 109). Shendure teaches a method wherein “All libraries were sequenced” (Para. 213), “were aligned to the human reference” (Para. 214), and “As read starts, we use both outer alignment coordinates of PE data for which both reads aligned to the same chromosome and where reads have opposite orientations. In cases where PE data was converted to single read data by adapter trimming, we consider both end coordinates of the SR alignment as read starts. For coverage, we consider all positions between the two (inferred) molecule ends, including these end positions. We define windowed protection scores (WPS) of a window size k as the number of molecules spanning a window minus those starting at any bases encompassed by the window.” (Para. 217).
Regarding claim 1, Shendure teaches “quantitation of… select samples with high burden of circulating tumor DNA” (Para. 75) and “cfDNA was screened for evidence of high tumor burden, along with total cfDNA yield” (Para. 201).
Regarding claim 1, Shendure teaches a method wherein “comprises a biological feature corresponding to quantitative expression of one or more genes.” (Para. 109) and “In some embodiments, the orthogonal biological feature is associated with highly expressed genes. In some embodiments, the orthogonal biological feature is associated with lowly expression genes.” (Para. 120).
Thus, Shendure suggests method comprising: determining in a blood sample obtained from a healthy subject and the diseased / tumor-bearing subject, a level of cell-free DNA (cfDNA) that maps to six or more nucleosome-depleted regions (NDR) comprising; sequencing cfDNA fragments in the blood sample to obtain sequencing reads; and determining the number of sequencing reads that align with the six or more NDRs to obtain said level of cfDNA that maps to the six or more NDRs; and quantifying the ctDNA burden based on said level of cfDNA using a model, wherein said NDRs (i) comprises the six or more NDRs of a-six or more genes which transcripts are differentially expressed between a healthy blood sample and a tumor blood sample.
Shendure does not explicitly teach the limitation “wherein the one or more NDR comprises one or more NDR of a gene selected from the group consisting of: SLC11A1, NLRP12, PRTN3, HMBS, LILRB3, ACSL1, GP9, MX2, RASGRP4, ATG16L2 and combinations thereof”.
Diehn discloses “Methods for creating a selector of mutated genomic regions and for using the selector set to analyze genetic alterations in a cell-free nucleic acid sample are provided. The methods can be used to measure tumor-derived nucleic acids in a blood sample from a subject and thus to monitor the progression of disease in the subject. The methods can also be used for cancer screening, cancer diagnosis, cancer prognosis, and cancer therapy designation.” (Abstract).
Regarding claim 1, Diehn teaches method comprising “The genomic regions may comprise genes, exonic regions, intronic regions, untranslated regions, non-coding regions or a combination thereof. The genomic regions may comprise two or more of exonic regions, intronic regions, and untranslated regions” (Para. 47; Para. 38), “selector set may comprise 1, 2, 3, 4,
5, 6, 7, 8, 9, 10 … or more genomic regions selected from Table 11” (Para. 52) and “NLRP12” (Pg. 342 Table 11-NLRP12). Furthermore, Diehn teaches a method comprising “methods of assessing tumor burden in a subject in need thereof. The method may comprise (a) obtaining sequence information on cell-free nucleic acids derived from a sample from the subject; (b) … determine quantities of circulating tumor DNA (ctDNA) in the sample; (c) assessing tumor burden based on the quantities of ctDNA”(Para. 118). “genomic regions may comprise genes, exonic regions, intronic regions, untranslated regions, non-coding regions or a combination thereof” read on NDRs.
Huang discloses “Molecular analysis of cell-free DNA (cfDNA) that circulates in plasma and other body fluids represents a “liquid biopsy” approach for non-invasive cancer screening or monitoring. The rapid development of sequencing technologies has made cfDNA a promising source to study cancer development and progression. Specific genetic and epigenetic alterations have been found in plasma, serum, and urine cfDNA and could potentially be used as diagnostic or prognostic biomarkers in various cancer types. In this review, we will discuss the molecular characteristics of cancer cfDNA and major bioinformatics approaches involved in the analysis of cfDNA sequencing data for detecting genetic mutation, copy number alteration, methylation change, and nucleosome positioning variation. We highlight specific challenges in sensitivity to detect genetic aberrations and robustness of statistical analysis. Finally, we provide perspectives regarding the standard and continuing development of bioinformatics analysis to move this promising screening tool into clinical practice.” (Abstract).
Regarding claim 1, Huang teaches method wherein “In addition to DNA methylation, cfDNA fragmentation and/or nucleosome occupancy patterns are another epigenetic feature to trace gene activity and tissue origin.” (Pg. 8, 7. Association of Nucleosome and Fragmentation Pattern with Tissue of Origin in cfDNA, Para. 1) and “Based on the expectation that fragment endpoints should cluster next to nucleosome boundaries and should be depleted at sites of nucleosome occupancy, Snyder et al. showed that nucleosome spacing patterns can inform the cell type of origin from cfDNA. The study showed that nucleosome spacing inferred from cfDNA in healthy individuals correlated strongly with epigenetic features of lymphoid and myeloid cells, consistent with hematopoietic cell death as a major source of cfDNA, while the patterns of nucleosome spacing in late-stage cancer patients match the anatomical origin of the patient’s cancer. Therefore, different nucleosome footprints between the tumor and the normal source of cfDNA may enable the noninvasive monitoring of a much broader set of clinical conditions than currently possible” Pg. 8, 7. Association of Nucleosome and Fragmentation Pattern with Tissue of Origin in cfDNA, Para. 2). Huang also teaches method wherein “By using TCGA Infinium HumanMethylation450 microarray data from both normal and tumor samples, CancerLocator identified as feature input a large number of CpG clusters that have high inter-individual methylation variation across all normal and cancer types. Since cfDNA from the peripheral blood is a mixture of normal and tumor DNA if a cancer cell is present, the methylation level for each CpG cluster, one for normal and the other one for a cancer type, can be estimated and the ctDNA fraction and the likelihood of the presence of a specific cancer type can be inferred based on the methylation data of informative CpG clusters” (Pg. 7-8, 7. Association of Nucleosome and Fragmentation Pattern with Tissue of Origin in cfDNA, Para. 3). “HumanMethylation450 microarray” reads on six or more NDRs of six or more genes selected from the group consisting of SLC11A1, NLRP12, PRTN3, HMBS, LILRB3, ACSL1,GP9, MX2, RASGRP4, ATG16L2 and combinations thereof. Thus, Shendure, Diehn, and Huang suggest a method wherein the six or more NDRs comprises six or more NDRs of a genes selected from the group consisting of: SLC11A1, NLRP12, PRTN3, HMBS, LILRB3, ACSL1,GP9, MX2, RASGRP4, ATG16L2 and combinations thereof.
Shendure, Diehn and Huang are considered to be analogous to the claimed invention because they are in the same field of using cfDNA to diagnose and/ monitor disease progression. Therefore, it would have been obvious to someone of ordinary skill in the art before the effective filing date of the claimed invention to have modified the method of quantifying ctDNA burden in the diseased/tumor-bearing subject as taught by Shendure to incorporate a method wherein six or more of the genomic regions selected comprises NLRP12 as taught by Diehn; and to incorporate a method wherein six or more of the genomic regions selected is SLC11A1, NLRP12, PRTN3, HMBS, LILRB3, ACSL1,GP9, MX2, RASGRP4, ATG16L2 and combinations thereof as taught by Huang and provide a method according to claim 1. Doing so would enable the prediction of tumor derived cfDNA in a tumor bearing subject.
The teachings of Shendure, Diehn and Huang are documented above in the rejection of claims 1 under 35 U.S.C. 103. Claims 3-11 and 13-15 depend on claim 1.
Regarding claim 3, Shendure teaches a method wherein “single-stranded library preparation protocol for cfDNA fragments” (Para. 34) and “The reaction was supplemented with biotinconjugated adapter oligo” (Para. 212). Furthermore, Diehn teaches a method wherein “Selecting the cfNA may comprise (i) hybridizing the cell-free nucleic acid sample to a plurality of selector set probes comprising a specific binding member; (ii) binding hybridized nucleic acids to a complementary specific binding member“(Para. 21). Thus, Shendure, Diehn and Huang suggest a method further comprising contacting the blood sample with six or more probes capable of binding to the six or more NDRs to capture cfDNA fragments comprising the six or more NDRs prior to the sequencing.
Regarding claim 4, Shendure teaches a method wherein “transcriptional start sites and exon-intron boundaries” (Para. 188). Thus, Shendure, Diehn and Huang suggest a method wherein the NDRs are selected from the group consisting of: a promoter region, a first exon-intron junction and combinations thereof.
Regarding claim 5, Shendure teaches a method wherein “a portion of cfDNA in bodily fluids such as circulating plasma can be derived from the tumor. The methods described here can potentially be used to detect and quantify this tumor derived portion” (Para. 126). Shendure teaches a method wherein “In some embodiments, the orthogonal biological feature is associated with highly expressed genes. In some embodiments, the orthogonal biological feature is associated with lowly expression genes.” (Para. 119). Thus, Shendure, Diehn and Huang suggest a method wherein the quantified ctDNA burden is associated /correlated with the level of cfDNA that maps to the six or more NDRs, wherein the association consists of positive and/or negative, linear and/or non-linear and monotonic and/or non-monotonic associations, optionally the quantified ctDNA burden is positively associated with a tumor burden in the diseased /tumor-bearing subject.
Regarding claim 6, Shendure teaches a method wherein “FPKM expression values, measured for 20,344 Ensembl gene identifiers” (Para. 226). Shendure teaches a method wherein “Table 5 tabulates sequencing-related statistics, including the total number of fragments sequenced, read lengths, the percentage of such fragments aligning to the reference with and without a mapping quality threshold, mean coverage, duplication rate, and the proportion of sequenced fragments in two length bins, for each sample. Fragment length was inferred from alignment of paired-end reads.” (Para. 204; Table 5). Shendure teaches a method wherein “When the signal was stratified based on gene expression in a lymphoid lineage cell line, NB-4, strong differences in the locations or intensity of nucleosome protection in relation to the TSS were observed, in highly vs. lowly expressed genes (FIG. 57).” (Para. 193). Thus, Shendure, Diehn and Huang suggest a method herein said transcript that is differentially expressed between the healthy blood sample and the tumor blood sample comprises a transcript which FPKM (Fragments Per Kilobase of transcript per Million),or RPKM (Reads Per Kilobase Million), or TPM (Transcripts Per Kilobase Million) value differs between the healthy blood sample and the tumor blood sample, optionally wherein the value differs by at least 10 times between the healthy blood sample and the tumor blood sample.
Regarding claim 7, Shendure teaches Table 5 which indicates different percentages of fragment lengths and different sequencing coverage for diseased samples (IC15, IC17, IC20, IC35, IC37 samples compared to Table 1 for healthy samples (IH02). (Table 5; Para. 177). Thus, Shendure, Diehn and Huang suggest a method wherein said NDRs that are degraded to different extents between cfDNA in the healthy blood sample and cfDNA in the blood sample of the diseased/tumor-bearing subject comprises-a NDRs having different sequencing coverage in the healthy blood sample and in the tumor blood sample.
Regarding claims 8-9, Shendure teaches a method wherein “wherein the reference map comprises a DNase I hypersensitive site dataset, an RNA expression dataset, expression data, a chromosome conformation map, a chromatin accessibility map, chromatin fragmentation map, or sequence data obtained from samples obtained from at least one reference subject, and corresponding to at least one cell type or tissue that is associated with a disease or a disorder, and/or positions or spacing of nucleosomes and/or chromatosomes in a tissue or cell type” (Claim 54). Shendure teaches a method wherein “the proportion assigned to each of the one or more determined tissues or cell types is based at least in part on a degree of correlation or of increased correlation, relative to cfDNA from a healthy subject or subjects” (Example 115). Shendure teaches a method wherein “In some embodiments, the orthogonal biological feature is associated with highly expressed genes. In some embodiments, the orthogonal biological feature is associated with lowly expression genes.” (Para. 99). Shendure teaches a method wherein “(an acute promyelocytic leukemia cell line) reveals differences in the spacing and placement of nucleosomes. Highly expressed genes show a strong phasing of nucleosomes within the transcript body.” Para. 70). Thus, Shendure, Diehn and Huang suggest a method wherein said transcript that is differentially expressed in the healthy blood sample and the tumor blood sample is selected from the group consisting of: a transcript that is more highly expressed in the healthy blood sample than in the tumor blood sample, a transcript that is more highly expressed in the tumor blood sample than in the healthy blood sample and combinations thereof; and wherein said transcript which is differentially expressed between the healthy blood sample and the tumor blood sample consists of transcript(s) that is more highly expressed in the healthy blood sample than in the tumor blood sample.
Regarding claim 10, Diehn teaches a method wherein “The plurality of regions… may comprise at least 5, 10… different genomic regions” (Para. 25) and “six or more regions may be indicative of an outcome of the cancer (Para. 102). Thus, Shendure, Diehn and Huang suggest a method wherein the six or more NDR comprises six NDRs, further optionally ten NDRs.
Regarding claim 11, Diehn teaches a method wherein “The total size of the plurality of genomic regions of the selector set may comprise less than… 30, 20, 10 or 5 kb of a genome.” (Para. 397). Thus, Shendure, Diehn and Huang suggest a method wherein the total length of the six or more NDRs is no more than 30 kb.
Regarding claim 13, Shendure further teaches a method wherein “the methods described herein are used for detection, monitoring and tissue(s) and/or cell-type(s)-of-origin assessment of malignancies from analysis of cfDNA in bodily fluids. It is now well documented that in patients with malignancies, a portion of cfDNA in bodily fluids such as circulating plasma can be derived from the tumor. The methods described here can potentially be used to detect and quantify this tumor derived portion.” (Para. 126) and “This may enable diagnosis and/or monitoring of pathological processes” (Para. 127). Diehn teaches a method wherein “In some embodiments, the ctDNA content in an individual' s blood, or blood derivative, sample is determined at one or more time points, optionally in conjunction with a therapeutic regimen. The presence of the ctDNA correlates with tumor burden, and is useful in monitoring response to therapy, monitoring residual disease, monitoring for the presence of metastases, monitoring total tumor burden, and the like” (Para. 16). Thus, Shendure, Diehn and Huang suggest a method wherein the method further comprises determining disease progression in the diseased /tumor-bearing subject comprising: determining in a subsequent blood sample obtained from the diseased / tumor- bearing subject, a level of cfDNA that maps to six or more NDRs; quantifying the ctDNA burden based on said level of cfDNA; comparing the ctDNA burden quantified from said subsequent blood sample with the ctDNA burden quantified from said blood sample; and identifying the diseased /tumor-bearing subject as having disease progression if the ctDNA burden quantified from said subsequent blood sample is higher than the ctDNA burden quantified from said blood sample and if the ctDNA burden quantified from said subsequent blood sample is not higher than the ctDNA burden quantified from said blood sample, the disease is identified to be improving/ablating in the diseased /tumor-bearing subject; wherein disease progression in the diseased /tumor-bearing subject indicates resistance to a treatment regimen received by the diseased /tumor-bearing subject.
Regarding claim 14, Shendure teaches a method wherein “In some embodiments, the report further includes a recommended treatment protocol including, for example and without limitation, a suggestion to obtain an additional diagnostic test from the subject, a suggestion to begin a therapeutic regimen, a suggestion to modify an existing therapeutic regimen with the subject, and/or a suggestion to suspend or stop an existing therapeutic regiment.” (Para. 103). Thus, Shendure, Diehn and Huang suggest a method further comprising changing the treatment regimen received by the diseased /tumor-bearing subject if the diseased /tumor-bearing subject is identified as having disease progression.
Regarding claim 15, Shendure teaches a method wherein “(colorectal adenocarcinoma” (Para. 74; Para. 203). Shendure teaches a method wherein “In some embodiments, the tissue or cell type is a biopsy from a tumor” (Para. 95). “colorectal adenocarcinoma” reads on colorectal tumor. Thus, Shendure, Diehn and Huang suggest a method wherein the tumor-bearing subject bears a colorectal tumor.
Response to Arguments
Applicant' s arguments filed 01/13/2026 (Pg.13-15) with respect to claim 1, 3-11 and 13-15 have been considered but are moot because the arguments filed 07/21/2026 do not apply to the new grounds of rejections. To clarify some instances argued in the response filed 01/13/2026 see responses to each argument made by Applicant below:
Applicants’ argument: “Shendure does not disclose a method of quantifying a ctDNA burden in a subject using cfDNA levels that map to six or more NDRs of the genes of the present claims. In detail, Shendure analyzed cfDNA coverage patterns at gene promoters but does not aim to, nor does it naturally lead to a method of quantifying a ctDNA burden / fraction” (Pg. 14)
Response: Applicant's arguments filed 01/13/2026 have been fully considered but they are not persuasive. As stated above in the new grounds of rejection, Shendure teaches “quantitation of… select samples with high burden of circulating tumor DNA” (Para. 75) and “cfDNA was screened for evidence of high tumor burden, along with total cfDNA yield” (Para. 201). Thus, Shendure does suggest a method of quantifying a ctDNA burden / fraction. Furthermore, as necessitated by amendment, see revised rejection towards claim 1 under 35 U.S.C. 103.
Applicants’ argument: “Diehn does not disclose a method of quantifying a ctDNA burden in a subject using cfDNA levels that map to six or more NDRs of the genes of the present claims..” (Pg. 13)
Response: Applicant's arguments filed 01/13/2026 have been fully considered but they are not persuasive. As stated above in the new grounds of rejection, Diehn teaches method comprising “The genomic regions may comprise genes, exonic regions, intronic regions, untranslated regions, non-coding regions or a combination thereof. The genomic regions may comprise two or more of exonic regions, intronic regions, and untranslated regions” (Para. 47; Para. 38), “selector set may comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 … or more genomic regions” (Para. 52). Thus, Shendure does suggest a method of quantifying a ctDNA burden in a subject using cfDNA levels that map to six or more NDRs of the genes. Furthermore, Diehn teaches a method comprising “methods of assessing tumor burden in a subject in need thereof. The method may comprise (a) obtaining sequence information on cell-free nucleic acids derived from a sample from the subject; (b) … determine quantities of circulating tumor DNA (ctDNA) in the sample; (c) assessing tumor burden based on the quantities of ctDNA”(Para. 118).
Conclusion of Response to Arguments
In view of the amendments, revised and new grounds of rejections, and responses to arguments are documented in this Final Office Action. No claims are in condition for allowance.
Conclusion
Applicant's amendment necessitated the new ground(s) of rejection presented in this Office action. Accordingly, THIS ACTION IS MADE FINAL. See MPEP § 706.07(a). Applicant is reminded of the extension of time policy as set forth in 37 CFR 1.136(a).
A shortened statutory period for reply to this final action is set to expire THREE MONTHS from the mailing date of this action. In the event a first reply is filed within TWO MONTHS of the mailing date of this final action and the advisory action is not mailed until after the end of the THREE-MONTH shortened statutory period, then the shortened statutory period will expire on the date the advisory action is mailed, and any nonprovisional extension fee (37 CFR 1.17(a)) pursuant to 37 CFR 1.136(a) will be calculated from the mailing date of the advisory action. In no event, however, will the statutory period for reply expire later than SIX MONTHS from the mailing date of this final action.
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/KENDRA R VANN-OJUEKAIYE/Examiner, Art Unit 1682
/WU CHENG W SHEN/Supervisory Patent Examiner, Art Unit 1682
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