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 .
The preliminary amendments dated 5/13/2024 are under consideration.
Priority
The present application is a 371 national stage entry of PCT/US2022/079735 (filed 11/11/2022), which claims benefit of US provisional application 63/278,921 (filed 11/12/2021).
Priority is recognized.
Information Disclosure Statement
The listing of references in the specification or the citation of references throughout the specification is not a proper information disclosure statement. 37 CFR 1.98(b) requires a list of all patents, publications, or other information submitted for consideration by the Office, and MPEP § 609.04(a) states, "the list may not be incorporated into the specification but must be submitted in a separate paper." Therefore, unless the references have been cited by the examiner on form PTO-892, they have not been considered.
Specification
The abstract of the disclosure is objected to because two copies have been filed, one on 5/13/2024 and one on 11/25/2024. There is no indication that the 11/25/2024 abstract is intended to replace the 5/13/2024 abstract in the file. A corrected abstract of the disclosure is required and must be presented on a separate sheet, apart from any other text. See MPEP § 608.01(b).
The disclosure is objected to because it contains an embedded hyperlink and/or other form of browser-executable code. Applicant is required to delete the embedded hyperlink and/or other form of browser-executable code; references to websites should be limited to the top-level domain name without any prefix such as http:// or other browser-executable code. See MPEP § 608.01. The embedded hyperlink appears in paragraph 74 on page 24.
The marked up disclosure is objected to because of the following informalities: the marked up disclosure includes part V after the “References”, which is the “ABSTRACT OF THE DISCLOSURE”. The original specification does not include this section and addition is not underlined.
Appropriate correction is required.
The use of terms that are trade names or marks used in commerce has been noted in this application. The terms should be accompanied by the generic terminology; furthermore the terms should be capitalized wherever they appear or, where appropriate, include a proper symbol indicating use in commerce such as ™, SM , or ® following the term.
Although the use of trade names and marks used in commerce (i.e., trademarks, service marks, certification marks, and collective marks) are permissible in patent applications, the proprietary nature of the marks should be respected and every effort made to prevent their use in any manner which might adversely affect their validity as commercial marks.
Claim Objections
Claim 1 is objected to because of the following informalities: the claim lacks punctuation after “comprising” in line 1 (e.g., said method comprising: a) …); the claim lacks articles prior to certain terms (e.g., “a methylation cfDNA library” starting in line 6, “sequencing an enriched methylated cfDNA library”, etc.). Appropriate correction is required.
Claim 2 is objected to because of the following informalities: the claim lacks punctuation after “comprising” in line 1 (e.g., said method comprising: a) …); the claim lacks articles prior to certain terms (e.g., “a methylation cfDNA library” starting in line 6, “sequencing an enriched methylated cfDNA library”, etc.). Appropriate correction is required.
Claim 3 is objected to because of the following informalities: the claim lacks punctuation after “comprising” in line 1 (e.g., said method comprising: a) …); the claim lacks articles prior to certain terms (e.g., “a methylation cfDNA library” starting in line 6, “sequencing an enriched methylated cfDNA library”, etc.). Appropriate correction is required.
Claim 5 is objected to because of the following informalities: the claim lacks punctuation after “comprising” in line 1 (e.g., said method comprising: a) …); the claim lacks articles prior to certain terms (e.g., “a methylation cfDNA library” starting in line 6, “sequencing an enriched methylated cfDNA library”, etc.). Appropriate correction is required.
Claim Interpretation
The independent claims refer to positions within the genome. The positions are interpreted in view of the specification as referring to the hg19 reference genome, which is also known in the field as GRCh37.
The independent claims recite a series of steps. MPEP 2111.01(II) states it is improper to read a specific order of steps into method claims where, as a matter of logic or grammar, the language of the method claims did not impose a specific order on the performance of the method steps, and the specification did not directly or implicitly require a particular order. Here, the independent claims do not necessarily imply all the steps are performed in the order recited. For example, step (b) of claim 1 is “extracting cfDNA”. The step does not specify from where the cfDNA is extracted and is not limited to extracting cfDNA from the obtained fluid biological sample. Step (f) is drawn to “enriching for methylated cfDNA”. The step does not specify from where the methylated cfDNA is being enriched and is not limited to the extracted cfDNA of step (b) or the ligated cfDNA of step (d). In step (f), the claim states “sequencing enriched methylated cfDNA library”. The step does not specify the step (e) is performed on the “methylation cfDNA library” of step (d) and is not limited as such. On the contrary, “the cfDNA” subjected to ligating in step (d) is interpreted as being that which was extracted in step (b) as it logically refers back to it.
The other independent claims use substantially the same claim language.
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, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 and 20 are rejected under 35 U.S.C. 101 because the claimed invention is directed to judicial exceptions without significantly more.
The claim(s) recite(s):
“assaying CpG islands for hypermethylation relative to a normal control”; and
“wherein the presence of CpG hypermethylation at a CpG islands…indicates…”.
The “assaying” step encompasses an abstract idea as it involves assaying for “hypermethylation relative to a normal control” and thus, involves comparing the methylation observed to that of a “normal control”. The limited amount of data may be considered mentally or with the aid of pen and paper.
The “wherein” clauses set forth natural correlations between the hypermethylation of particular genomic regions and a cancer.
The judicial exceptions are not integrated into a practical application because the claims do not involve:
improvements to the functioning of a computer or to any other technology or technical field;
applying or using the judicial exceptions to effect a particular treatment or prophylaxis for a disease or medical condition;
applying the judicial exception with, or by use of, a particular machine; or
effecting a transformation or reduction of a particular article to a different state or thing.
The claimed limitations add insignificant extra-solution activity to the judicial exceptions as they are data gather steps. While claim 1 further comprises “treating the cancer with an effective amount of a therapeutic agent”, the step is generic and is not based on observing any level of methylation. Thus, there is no nexus between the above judicial exceptions and the treating step of claim 1.
The claim(s) does/do not include additional elements that are sufficient to amount to significantly more than the judicial exception because the claims encompass the use of known assays, e.g., cfMBD-seq, as described in the instant specification (para. 119, p. 55).
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.
The following is a quotation of the first paragraph of pre-AIA 35 U.S.C. 112:
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 of carrying out his invention.
Claims 1-20 are rejected under 35 U.S.C. 112(a) or 35 U.S.C. 112 (pre-AIA ), first paragraph.
The specification, while being enabling for making determinations for the particular cancers based on the hypermethylation of the particular genomic regions listed in Table 1, does not reasonably provide enablement for making any determinations for any type of cancer based on hypermethylation of the particular genomic regions in the claims. The specification does not enable any person skilled in the art to which it pertains, or with which it is most nearly connected, to practice the invention commensurate in scope with these claims.
Claims broadly encompass indicating the presence of any type of cancer based on the hypermethylation of the genomic regions recited in the claims. The instant specification demonstrates that the recited genomic regions are associated with a particular type of cancer. For example, chr2:29337984-29338909 is associated with colorectal cancer and chr2:66672432-66673636 is associated with lung cancer. There is no indication that chr2:29337984-29338909 is associated with lung cancer, pancreatic cancer, breast cancer, liver cancer, skin cancer, etc.
Based on applicants analysis of methylation date, it is unreasonable to predict that the hypermethylation of the identified genomic regions can be used in the context of any other cancers other than those specifically identified in Table 1. It would require undue experimentation to collect and analyze additional methylation data from additional types of cancers with no predictable result, as the instant specification demonstrates that hypermethylation of those regions within cfDNA is specific for particular types of cancers.
Claim Rejections - 35 USC § 112
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 1-20 are rejected under 35 U.S.C. 112(b) or 35 U.S.C. 112 (pre-AIA ), second paragraph, as being indefinite for failing to particularly point out and distinctly claim the subject matter which the inventor or a joint inventor (or for applications subject to pre-AIA 35 U.S.C. 112, the applicant), regards as the invention.
Regarding claim 1, the claim recites “filler DNA” in line 5. The recitation lacks proper antecedent basis as “methylated filler DNA” was generated in step (c) and it is unclear if the step encompasses “combining” the ligated cfDNA with any generic “filler DNA”.
Regarding claim 2, the claim recites “filler DNA” in line 5. The recitation lacks proper antecedent basis as “methylated filler DNA” was generated in step (c) and it is unclear if the step encompasses “combining” the ligated cfDNA with any generic “filler DNA”.
Regarding claim 3, the claim recites “filler DNA” in line 5. The recitation lacks proper antecedent basis as “methylated filler DNA” was generated in step (c) and it is unclear if the step encompasses “combining” the ligated cfDNA with any generic “filler DNA”.
Regarding claim 5, the claim recites “filler DNA” in line 5. The recitation lacks proper antecedent basis as “methylated filler DNA” was generated in step (c) and it is unclear if the step encompasses “combining” the ligated cfDNA with any generic “filler DNA”.
The dependent claims all depend from claims 1, 2, 3 or 5 and are rejected for the same reason.
Claim Rejections - 35 USC § 102
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 the appropriate paragraphs of 35 U.S.C. 102 that form the basis for the rejections under this section made in this Office action:
A person shall be entitled to a patent unless –
(a)(1) the claimed invention was patented, described in a printed publication, or in public use, on sale, or otherwise available to the public before the effective filing date of the claimed invention.
(a)(2) the claimed invention was described in a patent issued under section 151, or in an application for patent published or deemed published under section 122(b), in which the patent or application, as the case may be, names another inventor and was effectively filed before the effective filing date of the claimed invention.
Claim(s) 1-20 is/are rejected under 35 U.S.C. 102(a)(1) as being anticipated by Shen (Nature. 2018. 563:579-583 (cited on the 3/13/2025 IDS) and Methods, Extended Data and Supplementary Tables).
Regarding claim 1, Shen teaches obtaining plasma as a “fluid biological sample” (Fig. 2; and Methods, Specimen processing for patient cfDNA).
Shen teaches extracting cfDNA from the plasma (Methods, Specimen processing for patient cfDNA).
Shen teaches generating “methylated filler DNA” by forming a mixture of unmethylated and in vitro methylated lambda amplicons generated using described primers (Methods, cfMeDIP-seq; and Supplementary Table 6).
Shen teaches ligating adapters to the cfDNA and combining it with the filler DNA (Methods, cfMeDIP-seq).
Shen teaches enriching for methylated DNA in the combination of ligated cfDNA and filler DNA using an antibody (Methods, cfMeDIP-seq).
Shen teaches amplifying and sequencing the methylation enriched cfDNA (Methods, cfMeDIP-seq).
Shen teaches assaying the sequencing data, including CpG islands, for hypermethylation relative to a normal control (Methods, Calculation and visualization of differentially methylated regions from cfDNA
of patients with pancreatic cancer and healthy donors; and Supplementary Table 1).
Shen further teaches the cancer of the patients was treated with surgical resection.
The claim includes a “wherein” clause stating the presence of hypermethylation within particular genomic regions is indicative of the presence of a cancer. The clause does not specify an active methods step of “indicating” the presence of a cancer based on observing hypermethylation; nor does the claim as a whole require detecting the hypermethylation within particular regions. Step (g) broadly encompasses simply comparing the methylation of any CpG islands to a normal control and detecting hypermethylation, hypomethylation, no methylation, etc. The “wherein” clause is interpreted as providing information relative to an intended use of the data gathered by the positively recited, active method steps of claim 1 and does not limit the scope of claim as a whole.
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It is noted that Shen does teach detecting hypermethylation of the following genomic locations that are encompassed by the present claims (Supplementary Table 1):
Regarding claim 2, Shen teaches obtaining plasma as a “fluid biological sample” (Fig. 2; and Methods, Specimen processing for patient cfDNA).
Shen teaches extracting cfDNA from the plasma (Methods, Specimen processing for patient cfDNA).
Shen teaches generating “methylated filler DNA” by forming a mixture of unmethylated and in vitro methylated lambda amplicons generated using described primers (Methods, cfMeDIP-seq; and Supplementary Table 6).
Shen teaches ligating adapters to the cfDNA and combining it with the filler DNA (Methods, cfMeDIP-seq).
Shen teaches enriching for methylated DNA within the combination of ligated cfDNA and filler DNA using an antibody (Methods, cfMeDIP-seq).
Shen teaches amplifying and sequencing the methylation enriched cfDNA (Methods, cfMeDIP-seq).
Shen teaches assaying the sequencing data, including CpG islands, for hypermethylation relative to a normal control (Methods, Calculation and visualization of differentially methylated regions from cfDNA
of patients with pancreatic cancer and healthy donors; and Supplementary Table 1).
The claim includes a “wherein” clause stating the presence of hypermethylation within particular genomic regions is indicative of the presence of a cancer. The clause does not specify an active methods step of “indicating” the presence of a cancer based on observing hypermethylation; nor does the claim as a whole require detecting the hypermethylation within particular regions. Step (g) broadly encompasses simply comparing the methylation of any CpG islands to a normal control and detecting hypermethylation, hypomethylation, no methylation, etc. The “wherein” clause is interpreted as providing information relative to an intended use of the data gathered by the positively recited, active method steps of claim 2 and does not limit the scope of claim as a whole.
It is noted that Shen does teach detecting hypermethylation of the above noted genomic locations that are encompassed by the present claims.
Regarding claim 3, Shen teaches obtaining plasma as a “fluid biological sample” (Fig. 2; and Methods, Specimen processing for patient cfDNA).
Shen teaches extracting cfDNA from the plasma (Methods, Specimen processing for patient cfDNA).
Shen teaches generating “methylated filler DNA” by forming a mixture of unmethylated and in vitro methylated lambda amplicons generated using described primers (Methods, cfMeDIP-seq; and Supplementary Table 6).
Shen teaches ligating adapters to the cfDNA and combining it with the filler DNA (Methods, cfMeDIP-seq).
Shen teaches enriching for methylated DNA in the combination of ligated cfDNA and filler DNA using an antibody (Methods, cfMeDIP-seq).
Shen teaches amplifying and sequencing the methylation enriched cfDNA (Methods, cfMeDIP-seq).
Shen teaches assaying the sequencing data, including CpG islands, for hypermethylation relative to a normal control (Methods, Calculation and visualization of differentially methylated regions from cfDNA
of patients with pancreatic cancer and healthy donors; and Supplementary Table 1).
The claim includes a “wherein” clause stating the presence of hypermethylation within particular genomic regions is indicative of the presence of a cancer. The clause does not specify an active methods step of “indicating” the presence of a cancer based on observing hypermethylation; nor does the claim as a whole require detecting the hypermethylation within particular regions. Step (g) broadly encompasses simply comparing the methylation of any CpG islands to a normal control and detecting hypermethylation, hypomethylation, no methylation, etc. The “wherein” clause is interpreted as providing information relative to an intended use of the data gathered by the positively recited, active method steps of claim 3 and does not limit the scope of claim as a whole.
It is noted that Shen does teach detecting hypermethylation of the above noted genomic locations that are encompassed by the present claims.
Regarding claim 4, the claim further describes the “cancer” set forth in the preamble. The positively recited, active method steps do not explicitly refer to cancer. MPEP 2111.02 states:
If the body of a claim fully and intrinsically sets forth all of the limitations of the claimed invention, and the preamble merely states, for example, the purpose or intended use of the invention, rather than any distinct definition of any of the claimed invention's limitations, then the preamble is not considered a limitation and is of no significance to claim construction.
Accordingly, the preamble merely sets forth the intended use or purpose of the claimed methods, but does not limit the scope of the claims.
It is noted that Shen teaches the patients have pancreatic cancer (Fig. 2).
Regarding claim 5, Shen teaches obtaining plasma as a “fluid biological sample” (Fig. 2; and Methods, Specimen processing for patient cfDNA).
Shen teaches extracting cfDNA from the plasma (Methods, Specimen processing for patient cfDNA).
Shen teaches generating “methylated filler DNA” by forming a mixture of unmethylated and in vitro methylated lambda amplicons generated using described primers (Methods, cfMeDIP-seq; and Supplementary Table 6).
Shen teaches ligating adapters to the cfDNA and combining it with the filler DNA (Methods, cfMeDIP-seq).
Shen teaches enriching for methylated DNA in the combination of ligated cfDNA and filler DNA using an antibody (Methods, cfMeDIP-seq).
Shen teaches amplifying and sequencing the methylation enriched cfDNA (Methods, cfMeDIP-seq).
Shen teaches assaying the sequencing data, including CpG islands, for hypermethylation relative to a normal control (Methods, Calculation and visualization of differentially methylated regions from cfDNA
of patients with pancreatic cancer and healthy donors; and Supplementary Table 1).
The claim includes a “wherein” clause stating the presence of hypermethylation within particular genomic regions is indicative of the presence of a cancer. The clause does not specify an active methods step of “indicating” the presence of a cancer based on observing hypermethylation; nor does the claim as a whole require detecting the hypermethylation within particular regions. Step (g) broadly encompasses simply comparing the methylation of any CpG islands to a normal control and detecting hypermethylation, hypomethylation, no methylation, etc. The “wherein” clause is interpreted as providing information relative to an intended use of the data gathered by the positively recited, active method steps of claim 3 and does not limit the scope of claim as a whole.
It is noted that Shen does teach detecting hypermethylation of the above noted genomic locations that are encompassed by the present claims.
Regarding claim 6, Shen teaches the “fluid biological sample” is plasma as described above.
Regarding claim 7, the “methylated filler DNA” of Shen has the same structure as that generated as described in claim 7 because it is amplified lambda DNA that has been methylated in vitro (Methods, cfMeDIP-seq).
Regarding claim 8, Shen teaches hypermethylation is determined relative to “a control standard” in the form of cell-free DNA from healthy controls or from normal tissue from the patient (Fig. 2).
Regarding claim 9, the claim further describes the “cancer” set forth in the preamble. The positively recited, active method steps do not explicitly refer to cancer. MPEP 2111.02 states:
If the body of a claim fully and intrinsically sets forth all of the limitations of the claimed invention, and the preamble merely states, for example, the purpose or intended use of the invention, rather than any distinct definition of any of the claimed invention's limitations, then the preamble is not considered a limitation and is of no significance to claim construction.
Accordingly, the preamble merely sets forth the intended use or purpose of the claimed methods, but does not limit the scope of the claims.
It is noted that Shen teaches the patients have pancreatic cancer (Fig. 2).
Regarding claim 10, Shen teaches the “fluid biological sample” is plasma as described above.
Regarding claim 11, the “methylated filler DNA” of Shen has the same structure as that generated as described in claim 11 because it is amplified lambda DNA that has been methylated in vitro (Methods, cfMeDIP-seq).
Regarding claim 12, Shen teaches hypermethylation is determined relative to “a control standard” in the form of cell-free DNA from healthy controls or from normal tissue from the patient (Fig. 2).
Regarding claim 13, the claim further describes the “cancer” set forth in the preamble. The positively recited, active method steps do not explicitly refer to cancer. MPEP 2111.02 states:
If the body of a claim fully and intrinsically sets forth all of the limitations of the claimed invention, and the preamble merely states, for example, the purpose or intended use of the invention, rather than any distinct definition of any of the claimed invention's limitations, then the preamble is not considered a limitation and is of no significance to claim construction.
Accordingly, the preamble merely sets forth the intended use or purpose of the claimed methods, but does not limit the scope of the claims.
It is noted that Shen teaches the patients have pancreatic cancer (Fig. 2).
Regarding claim 14, Shen teaches the “fluid biological sample” is plasma as described above.
Regarding claim 15, the “methylated filler DNA” of Shen has the same structure as that generated as described in claim 15 because it is amplified lambda DNA that has been methylated in vitro (Methods, cfMeDIP-seq).
Regarding claim 16, Shen teaches hypermethylation is determined relative to “a control standard” in the form of cell-free DNA from healthy controls or from normal tissue from the patient (Fig. 2).
Regarding claim 17, the claim further describes the “cancer” set forth in the preamble. The positively recited, active method steps do not explicitly refer to cancer. MPEP 2111.02 states:
If the body of a claim fully and intrinsically sets forth all of the limitations of the claimed invention, and the preamble merely states, for example, the purpose or intended use of the invention, rather than any distinct definition of any of the claimed invention's limitations, then the preamble is not considered a limitation and is of no significance to claim construction.
Accordingly, the preamble merely sets forth the intended use or purpose of the claimed methods, but does not limit the scope of the claims.
It is noted that Shen teaches the patients have pancreatic cancer (Fig. 2).
Regarding claim 18, Shen teaches the “fluid biological sample” is plasma as described above.
Regarding claim 19, the “methylated filler DNA” of Shen has the same structure as that generated as described in claim 15 because it is amplified lambda DNA that has been methylated in vitro (Methods, cfMeDIP-seq).
Regarding claim 20, Shen teaches hypermethylation is determined relative to “a control standard” in the form of cell-free DNA from healthy controls or from normal tissue from the patient (Fig. 2).
Claim(s) 1-20 is/are rejected under 35 U.S.C. 102(a)(1) as being anticipated by Huang (Cancers. 2021. 13:5611; cited on the 5/13/2024 IDS).
Regarding claims 1 and 6, Huang discloses a method of treating a cancer (abstract), said method comprising
a) obtaining a fluid biological sample (abstract);
b) extracting cfDNA (page 3, paragraphs 2-3);
c) generating methylated filler DNA (page 4, paragraph 2);
d) ligating an adapter to the cfDNA and combining with filler DNA thereby creating methylation cfDNA library comprised wherein adapter ligated cfDNA was first combined with methylated filler DNA (page 4, paragraph 3);
e) enriching for methylated cfDNA (page 4, paragraph 3);
f) amplifying and sequencing enriched methylated cfDNA library (page 4, paragraph 5);
g) assaying CpG islands for hypermethylation relative to a normal control (abstract);
wherein the presence of CpG hypermethylation at a CpG island (table 1 shows differentially methylated CgG islands linked to their genes, several genes with promoter hypermethylation; page 11, paragraph 2) chr4:174427892-174428192, chr7:27265159-27265493, chr7:65037625-65037864,chr8: 124172801-124173541, chr12:54408427-54408713, chr13:28549840-28550246, chr1 :50798668-50799536, chr5:92939796-92940216, or chr12:114881650-114881937, and/or at CpG island associated with CLIP4 chr2:29337984-29338909), LONRF2 chr2:100937780-100939059, RNF217 chr6: 125283125-125284389, MEIS1 chr2:66672432-66673636, ZNF638 chr2:71503548-71504233, WNT6 chr2:219736133-219736592, MGST2 chr4:140655963-140657135, PTGER4 chr5:40679503-40682081, C9orf129 chr9:96108467-96108992,B4GALNT1 chr12:58021295-58022037, HOXB8 chr17:46691521-46692097, TBX4 chr17:59539363-59539834, SOX9 chr17:70112825-70114271, RNF220 chr1 :44883137-44884272, CELF2 chr10: 11059443-11060524, and/or DBX1 chr11 :20177609-20178824 (Table 1 lists all of the chromosome sites disclosed by the PCT application, e.g., chr4:174427892-174428192, chr7:27265159-27265493, chr7:65037625-65037864, chr8: 124172801-124173541, chr12:54408427-54408713, chr13:28549840-28550246, chr1 :50798668-50799536, chr5:92939796-92940216, and chr12: 114881650-114881937 and CL! P4 (chr2:29337984-29338909), LONRF2 (chr2: 100937780-100939059), RNF217 (chr6: 125283125-125284389), MEIS1(chr2:66672432-66673636), ZNF638 (chr2:71503548-71504233), WNT6 (chr2:219736133-219736592), MGST2 (chr4:140655963:140657135), PTGER4 (chr5:40679503-40682081 ), C9orf129 (chr9:96108467-96108992),B4GALNT1 (chr12:58021295-58022037), HOXB8 (chr17:46691521-46692097), TBX4 (chr17:59539363-59539834), SOX9 (chr17:70112825-70114271), RNF220 (chr1:44883137-44884272), CELF2 (chr10:11059443-11060524), and DBX1 (chr11 :20177609-20178824); Table 1) indicates the presence of a cancer (Table 1), and
h) treating the cancer with an effective amount of a therapeutic agent (abstract).
Regarding claim 2 and 10, HUANG discloses a method of detecting a cancer (abstract), said method comprising
a) obtaining a fluid biological sample (abstract);
b) extracting cfDNA (page 3, paragraphs 2-3);
c) generating methylated filler DNA (page 4, paragraph 2);
d) ligating an adapter to the cfDNA and combining with filler DNA thereby creating methylation cfDNA library comprised wherein adapter ligated cfDNA was first combined with methylated filler DNA (page 4, paragraph 3);
e) enriching for methylated cfDNA (page 4, paragraph 3);
f) amplifying and sequencing enriched methylated cfDNA library (page 4, paragraph 5);
g) assaying CpG islands for hypermethylation relative to a normal control (abstract);
wherein the presence of CpG hypermethylation at a CpG island (table 1 shows differentially methylated CgG islands linked to their genes, several genes with promoter hypermethylation; page 11, paragraph 2) chr4:174427892-174428192, chr7:27265159-27265493, chr7:65037625-65037864,chr8: 124172801-124173541, chr12:54408427-54408713, chr13:28549840-28550246, chr1 :50798668-50799536, chr5:92939796-92940216, or chr12:114881650-114881937, and/or at CpG island associated with CLIP4 chr2:29337984-29338909), LONRF2 chr2:100937780-100939059, RNF217 chr6: 125283125-125284389, MEIS1 chr2:66672432-66673636, ZNF638 chr2:71503548-71504233, WNT6 chr2:219736133-219736592, MGST2 chr4:140655963-140657135, PTGER4 chr5:40679503-40682081, C9orf129 chr9:96108467-96108992,B4GALNT1 chr12:58021295-58022037, HOXB8 chr17:46691521-46692097, TBX4 chr17:59539363-59539834, SOX9 chr17:70112825-70114271, RNF220 chr1 :44883137-44884272, CELF2 chr10: 11059443-11060524, and/or DBX1 chr11 :20177609-20178824 (Table 1 lists all of the chromosome sites disclosed by the PCT application, e.g., chr4:174427892-174428192, chr7:27265159-27265493, chr7:65037625-65037864, chr8: 124172801-124173541, chr12:54408427-54408713, chr13:28549840-28550246, chr1 :50798668-50799536, chr5:92939796-92940216, and chr12: 114881650-114881937 and CL! P4 (chr2:29337984-29338909), LONRF2 (chr2: 100937780-100939059), RNF217 (chr6: 125283125-125284389), MEIS1(chr2:66672432-66673636), ZNF638 (chr2:71503548-71504233), WNT6 (chr2:219736133-219736592), MGST2 (chr4:140655963:140657135), PTGER4 (chr5:40679503-40682081 ), C9orf129 (chr9:96108467-96108992),B4GALNT1 (chr12:58021295-58022037), HOXB8 (chr17:46691521-46692097), TBX4 (chr17:59539363-59539834), SOX9 (chr17:70112825-70114271), RNF220 (chr1:44883137-44884272), CELF2 (chr10:11059443-11060524), and DBX1 (chr11 :20177609-20178824); Table 1) indicates the presence of a cancer (Table 1), and
h) treating the cancer with an effective amount of a therapeutic agent (abstract).
Regarding claim 3 and 14, HUANG discloses a method of grading a cancer (abstract), said method comprising
a) obtaining a fluid biological sample (abstract);
b) extracting cfDNA (page 3, paragraphs 2-3);
c) generating methylated filler DNA (page 4, paragraph 2);
d) ligating an adapter to the cfDNA and combining with filler DNA thereby creating methylation cfDNA library comprised wherein adapter ligated cfDNA was first combined with methylated filler DNA (page 4, paragraph 3);
e) enriching for methylated cfDNA (page 4, paragraph 3);
f) amplifying and sequencing enriched methylated cfDNA library (page 4, paragraph 5);
g) assaying CpG islands for hypermethylation relative to a normal control (abstract);
wherein the presence of CpG hypermethylation at a CpG island (table 1 shows differentially methylated CgG islands linked to their genes, several genes with promoter hypermethylation; page 11, paragraph 2) chr4:174427892-174428192, chr7:27265159-27265493, chr7:65037625-65037864,chr8: 124172801-124173541, chr12:54408427-54408713, chr13:28549840-28550246, chr1 :50798668-50799536, chr5:92939796-92940216, or chr12:114881650-114881937, and/or at CpG island associated with CLIP4 chr2:29337984-29338909), LONRF2 chr2:100937780-100939059, RNF217 chr6: 125283125-125284389, MEIS1 chr2:66672432-66673636, ZNF638 chr2:71503548-71504233, WNT6 chr2:219736133-219736592, MGST2 chr4:140655963-140657135, PTGER4 chr5:40679503-40682081, C9orf129 chr9:96108467-96108992,B4GALNT1 chr12:58021295-58022037, HOXB8 chr17:46691521-46692097, TBX4 chr17:59539363-59539834, SOX9 chr17:70112825-70114271, RNF220 chr1 :44883137-44884272, CELF2 chr10: 11059443-11060524, and/or DBX1 chr11 :20177609-20178824 (Table 1 lists all of the chromosome sites disclosed by the PCT application, e.g., chr4:174427892-174428192, chr7:27265159-27265493, chr7:65037625-65037864, chr8: 124172801-124173541, chr12:54408427-54408713, chr13:28549840-28550246, chr1 :50798668-50799536, chr5:92939796-92940216, and chr12: 114881650-114881937 and CL! P4 (chr2:29337984-29338909), LONRF2 (chr2: 100937780-100939059), RNF217 (chr6: 125283125-125284389), MEIS1(chr2:66672432-66673636), ZNF638 (chr2:71503548-71504233), WNT6 (chr2:219736133-219736592), MGST2 (chr4:140655963:140657135), PTGER4 (chr5:40679503-40682081 ), C9orf129 (chr9:96108467-96108992),B4GALNT1 (chr12:58021295-58022037), HOXB8 (chr17:46691521-46692097), TBX4 (chr17:59539363-59539834), SOX9 (chr17:70112825-70114271), RNF220 (chr1:44883137-44884272), CELF2 (chr10:11059443-11060524), and DBX1 (chr11 :20177609-20178824); Table 1) indicates the presence of a cancer (Table 1), and
h) treating the cancer with an effective amount of a therapeutic agent (abstract).
Regarding claim 4, Huang discloses the method of claim 2, and HUANG further discloses wherein the cancer is pancreatic, colorectal, or lung cancer (colorectal, lung, pancreatic cancer; page 3, paragraph 2; Table 1 ).
Regarding claim 5 and 18, Huang discloses a method of method of typing a cancer (page 11, paragraph 1 ), said method comprising
Huang discloses a method of treating a cancer (abstract), said method comprising
a) obtaining a fluid biological sample (abstract);
b) extracting cfDNA (page 3, paragraphs 2-3);
c) generating methylated filler DNA (page 4, paragraph 2);
d) ligating an adapter to the cfDNA and combining with filler DNA thereby creating methylation cfDNA library comprised wherein adapter ligated cfDNA was first combined with methylated filler DNA (page 4, paragraph 3);
e) enriching for methylated cfDNA (page 4, paragraph 3);
f) amplifying and sequencing enriched methylated cfDNA library (page 4, paragraph 5);
g) assaying CpG islands for hypermethylation relative to a normal control (abstract);
wherein the presence of CpG hypermethylation at a CpG island (table 1 shows differentially methylated CgG islands linked to their genes, several genes with promoter hypermethylation; page 11, paragraph 2) chr4:174427892-174428192, chr7:27265159-27265493, chr7:65037625-65037864,chr8: 124172801-124173541, chr12:54408427-54408713, chr13:28549840-28550246, chr1 :50798668-50799536, chr5:92939796-92940216, or chr12:114881650-114881937, and/or at CpG island associated with CLIP4 chr2:29337984-29338909), LONRF2 chr2:100937780-100939059, RNF217 chr6: 125283125-125284389, MEIS1 chr2:66672432-66673636, ZNF638 chr2:71503548-71504233, WNT6 chr2:219736133-219736592, MGST2 chr4:140655963-140657135, PTGER4 chr5:40679503-40682081, C9orf129 chr9:96108467-96108992,B4GALNT1 chr12:58021295-58022037, HOXB8 chr17:46691521-46692097, TBX4 chr17:59539363-59539834, SOX9 chr17:70112825-70114271, RNF220 chr1 :44883137-44884272, CELF2 chr10: 11059443-11060524, and/or DBX1 chr11 :20177609-20178824 (Table 1 lists all of the chromosome sites disclosed by the PCT application, e.g., chr4:174427892-174428192, chr7:27265159-27265493, chr7:65037625-65037864, chr8: 124172801-124173541, chr12:54408427-54408713, chr13:28549840-28550246, chr1 :50798668-50799536, chr5:92939796-92940216, and chr12: 114881650-114881937 and CL! P4 (chr2:29337984-29338909), LONRF2 (chr2: 100937780-100939059), RNF217 (chr6: 125283125-125284389), MEIS1(chr2:66672432-66673636), ZNF638 (chr2:71503548-71504233), WNT6 (chr2:219736133-219736592), MGST2 (chr4:140655963:140657135), PTGER4 (chr5:40679503-40682081 ), C9orf129 (chr9:96108467-96108992),B4GALNT1 (chr12:58021295-58022037), HOXB8 (chr17:46691521-46692097), TBX4 (chr17:59539363-59539834), SOX9 (chr17:70112825-70114271), RNF220 (chr1:44883137-44884272), CELF2 (chr10:11059443-11060524), and DBX1 (chr11 :20177609-20178824); Table 1) indicates the presence of a cancer (Table 1), and
Regarding claim 7, Huang teaches:
To generate filler DNA, enterobacteria phage _ DNA was polymerase chain reaction (PCR) amplified with GoTaq Master Mix (Promega, Madison, WI, USA). The primer sequences were as follows: forward primer 5’-CGATGGGTTAATTCGCTCGTTGTGG-3’, reverse primer 5’-GCACAACGGAAAGAGCACTG-3’. The 274-bp amplicons were treated with CpG methyltransferase (M.SssI, Thermo Fisher Scientific, Waltham, MA, USA) to methylate amplicons. Methylated amplicons were purified using a DNA Clean and Concentrator-5 Kit (ZYMO Research, Irvine, CA, USA) and quantified using a Qubit Fluorometer. CpG methylation-sensitive restriction enzyme HpyCH4IV (New England BioLabs, Ipswitch, MA, USA) digestion, followed by agarose gel electrophoresis, was performed to ensure the complete methylation of filler DNA. (page 4 of 18).
Regarding claim 8, Huang teaches the “normal control” is control versus a case (p. 5 of 18, 2.9. Differential Methylation Analysis of cfMBD-Seq Data and 2.10. Methylation Analyses for Tumor-Tissue-Specific DMCGIs).
Regarding claim 9, Huang discloses the method of claim 2, and Huang further discloses wherein the cancer is pancreatic, colorectal, or lung cancer (colorectal, lung, pancreatic cancer; page 3, paragraph 2; Table 1 ).
Regarding claim 11, Huang teaches:
To generate filler DNA, enterobacteria phage _ DNA was polymerase chain reaction (PCR) amplified with GoTaq Master Mix (Promega, Madison, WI, USA). The primer sequences were as follows: forward primer 5’-CGATGGGTTAATTCGCTCGTTGTGG-3’, reverse primer 5’-GCACAACGGAAAGAGCACTG-3’. The 274-bp amplicons were treated with CpG methyltransferase (M.SssI, Thermo Fisher Scientific, Waltham, MA, USA) to methylate amplicons. Methylated amplicons were purified using a DNA Clean and Concentrator-5 Kit (ZYMO Research, Irvine, CA, USA) and quantified using a Qubit Fluorometer. CpG methylation-sensitive restriction enzyme HpyCH4IV (New England BioLabs, Ipswitch, MA, USA) digestion, followed by agarose gel electrophoresis, was performed to ensure the complete methylation of filler DNA. (page 4 of 18).
Regarding claim 12, Huang teaches the “normal control” is control versus a case (p. 5 of 18, 2.9. Differential Methylation Analysis of cfMBD-Seq Data and 2.10. Methylation Analyses for Tumor-Tissue-Specific DMCGIs).
Regarding claim 13, Huang discloses the method of claim 2, and HUANG further discloses wherein the cancer is pancreatic, colorectal, or lung cancer (colorectal, lung, pancreatic cancer; page 3, paragraph 2; Table 1 ).
Regarding claim 15, Huang teaches:
To generate filler DNA, enterobacteria phage _ DNA was polymerase chain reaction (PCR) amplified with GoTaq Master Mix (Promega, Madison, WI, USA). The primer sequences were as follows: forward primer 5’-CGATGGGTTAATTCGCTCGTTGTGG-3’, reverse primer 5’-GCACAACGGAAAGAGCACTG-3’. The 274-bp amplicons were treated with CpG methyltransferase (M.SssI, Thermo Fisher Scientific, Waltham, MA, USA) to methylate amplicons. Methylated amplicons were purified using a DNA Clean and Concentrator-5 Kit (ZYMO Research, Irvine, CA, USA) and quantified using a Qubit Fluorometer. CpG methylation-sensitive restriction enzyme HpyCH4IV (New England BioLabs, Ipswitch, MA, USA) digestion, followed by agarose gel electrophoresis, was performed to ensure the complete methylation of filler DNA. (page 4 of 18).
Regarding claim 16, Huang teaches the “normal control” is control versus a case (p. 5 of 18, 2.9. Differential Methylation Analysis of cfMBD-Seq Data and 2.10. Methylation Analyses for Tumor-Tissue-Specific DMCGIs).
Regarding claim 17, Huang discloses the method of claim 2, and HUANG further discloses wherein the cancer is pancreatic, colorectal, or lung cancer (colorectal, lung, pancreatic cancer; page 3, paragraph 2; Table 1 ).
Regarding claim 19, Huang teaches:
To generate filler DNA, enterobacteria phage _ DNA was polymerase chain reaction (PCR) amplified with GoTaq Master Mix (Promega, Madison, WI, USA). The primer sequences were as follows: forward primer 5’-CGATGGGTTAATTCGCTCGTTGTGG-3’, reverse primer 5’-GCACAACGGAAAGAGCACTG-3’. The 274-bp amplicons were treated with CpG methyltransferase (M.SssI, Thermo Fisher Scientific, Waltham, MA, USA) to methylate amplicons. Methylated amplicons were purified using a DNA Clean and Concentrator-5 Kit (ZYMO Research, Irvine, CA, USA) and quantified using a Qubit Fluorometer. CpG methylation-sensitive restriction enzyme HpyCH4IV (New England BioLabs, Ipswitch, MA, USA) digestion, followed by agarose gel electrophoresis, was performed to ensure the complete methylation of filler DNA. (page 4 of 18).
Regarding claim 20, Huang teaches the “normal control” is control versus a case (p. 5 of 18, 2.9. Differential Methylation Analysis of cfMBD-Seq Data and 2.10. Methylation Analyses for Tumor-Tissue-Specific DMCGIs).
Conclusion
No claims allowed.
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/JOSEPH G. DAUNER/Primary Examiner, Art Unit 1682
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