DETAILED ACTION
The amendment filed on 01/28/2026 has been entered.
Claims 1 and 3-4 were amended in the claim set filed on 01/28/2026.
Applicant’s election without traverse of Group II, claims 1, 3, 4, and 8, drawn to a method for the diagnosis of human subjects at risk of developing advanced colorectal adenomas and species of miR15b and miR29a in the reply filed on 07/17/2025 is acknowledged.
Claims 6-8 are withdrawn drawn to a nonelected Group II and non-elected species.
Claims 11-20 were added in the claim set filed on 01/28/2026. No new matter was added.
Claims 1, 3-4 and 11-20 in the claim set filed on 01/28/2026 are currently under examination.
Response to the Arguments
Objections to the Drawings and Specification in the previously mailed non-final have been withdrawn in light of applicants Drawings and Specification amendments.
Applicant’s arguments regarding previous rejection(s) of claim(s) 1 and 3-4 under 35 U.S.C. 101 have been fully considered and are persuasive. Applicant' s argument on Pg. 12, states that “Because the feature (iii) of Claim 3 adds significantly more to the alleged judicial exception relating to the expression pattern or level of the recited microRNAs, Applicant respectfully requests withdrawal of the Section 101 rejection of Claim 3.” The 35 U.S.C. 101 rejections documented in the previously mailed non-final have been withdrawn in light of applicants claim amendments and arguments on Pg. 9-14. However, upon further consideration and search, new grounds of rejection for claims 1, 3-4 and 11-20 are made as documented below in the 35 U.S.C. 101 rejection in this office action on Pg. 4-14.
As necessitated by amendment, the 35 U.S.C. 103 rejections of claim(s) 1, 3-4 documented in the previously mailed non-final have been withdrawn in light of applicants claim amendments. However, upon further consideration and search, new grounds of rejection are made as documented below in the 35 U.S.C. 103 rejection in this office action on Pg. 17-36.
The objections and rejections for claims 1, 3-4 and 11-20 are documented below in this Final Office Action are necessitated by claim amendments filed on 01/28/2026.
Priority
This application is a CON of 16/464,674 (Abandoned on 01/31/2022), filed on 05/28/2019, which is a 371 of PCT/EP2017/080560, filed on 11/27/2017. Acknowledgment is made of applicant's claim for foreign priority based on an application filed in Europe on 11/28/2016. A certified copy of the EP16382567.2 application was filed on 05/28/2019 in parent application 16/464,674. Accordingly, The priority date of claim set filed on 01/28/2026 for instant application 17/570,221 is determined to be 11/28/2016, the filing date of EP16382567.2.
Claim Objections
Claim 1 is objected to because of the following informalities: “heathy’ should be “healthy”. Appropriate correction is required.
Claim Rejections - 35 USC § 112(d)
The following is a quotation of 35 U.S.C. 112(d):
(d) REFERENCE IN DEPENDENT FORMS.—Subject to subsection (e), a claim in dependent form shall contain a reference to a claim previously set forth and then specify a further limitation of the subject matter claimed. A claim in dependent form shall be construed to incorporate by reference all the limitations of the claim to which it refers.
The following is a quotation of pre-AIA 35 U.S.C. 112, fourth paragraph:
Subject to the following paragraph [i.e., the fifth paragraph of pre-AIA 35 U.S.C. 112], a claim in dependent form shall contain a reference to a claim previously set forth and then specify a further limitation of the subject matter claimed. A claim in dependent form shall be construed to incorporate by reference all the limitations of the claim to which it refers.
Claim 14 is rejected under 35 U.S.C. 112(d) or pre-AIA 35 U.S.C. 112, 4th paragraph, as being of improper dependent form for failing to further limit the subject matter of the claim upon which it depends, or for failing to include all the limitations of the claim upon which it depends. The limitation of claim 14 fails to further limit the subject matter of claim 13, which it depends on. Applicant may cancel the claim(s), amend the claim(s) to place the claim(s) in proper dependent form, rewrite the claim(s) in independent form, or present a sufficient showing that the dependent claim(s) complies with the statutory requirements.
Claim 20 is rejected under 35 U.S.C. 112(d) or pre-AIA 35 U.S.C. 112, 4th paragraph, as being of improper dependent form for failing to further limit the subject matter of the claim upon which it depends, or for failing to include all the limitations of the claim upon which it depends. The limitation of claim 20 fails to further limit the subject matter of claim 3, which it depends on. Applicant may cancel the claim(s), amend the claim(s) to place the claim(s) in proper dependent form, rewrite the claim(s) in independent form, or present a sufficient showing that the dependent claim(s) complies with the statutory requirements.
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, 3-4 and 11-20 are rejected under 35 U.S.C. 101 because the claimed invention is directed towards abstract ideas/mental process of obtaining, measuring, comparing, and diagnosing, abstract idea/mathematical concept of normalization based on a geometric mean, a natural correlation of expression pattern/level of miRNA to advanced colorectal adenoma and routine and conventional methods of obtaining sample, measuring expression, analysis normalization and/or administering a treatment, without significantly more. The claim(s) recite(s) 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.
Claim analysis
The instant claim 1 is directed towards: A method for treating advanced colorectal adenoma in a human subject, the method comprising: (a) obtaining a biological sample from the human subject; (b) measuring expression levels of at least miR-15b and miR-29a in the biological sample from the human subject; (c) comparing the expression levels of the miR-15b and the miR-29a in the biological sample from the human subject to expression levels of miR-15b and miR-29a in a corresponding biological sample from a healthy subject not suffering from advanced colorectal adenoma; (d) diagnosing the human subject with the advanced colorectal adenoma when both of the miR-15b and the miR-29a in the biological sample from the human subject are overexpressed relative to the expression levels of the miR-15b and the miR- 29a in the corresponding biological sample from the heathy subject; and (e) treating the human subject for the advanced colorectal adenoma by performing a surgical treatment, a chemotherapy treatment, an antibody treatment, an anti-vascular endothelial growth factor receptor (VEGF/R) treatment, a radiation treatment, or any combination thereof, on the human subject, wherein the biological sample comprises a blood sample, a plasma sample or a serum sample.
The steps (a) obtaining a biological sample … (b) measuring expression levels… (c) comparing the expression levels… in the biological sample from the human subject to expression levels of… in a corresponding biological sample from a healthy subject… (d) diagnosing the human subject with the advanced colorectal adenoma… are an abstract idea relating to organizing or analyzing information in a way that can be performed mentally or is analogous to human mental work and the steps are also considered routine and conventional as demonstrated by the 35 USC § 103 rejections stated below. (See MPEP 2106.04(a)(2) and 2106.05(d)).
The correlation between when both miR-15b and miR-29a in the biological sample from a human subject are overexpressed as compared to the expression levels in a healthy subject to advanced colorectal adenoma is based on a natural phenomenon, as the expression is naturally occurring in subjects with advanced colorectal adenoma. (See 2106.04(b)).
Although claim 1 step (d) recites treating the human subject for the advanced colorectal adenoma by performing a surgical treatment, a chemotherapy treatment, an antibody treatment, an anti-vascular endothelial growth factor receptor (VEGF/R) treatment, a radiation treatment, or any combination thereof, on the human subject, which is of great generality being routine and conventional as demonstrated in the prior art rejections documented below and is not a particular treatment.
Regarding claim 15, the claim recites: further comprising performing colonoscopy on the human subject to confirm that the human subject has the advanced colorectal adenoma, before treating of the human subject for the advanced colorectal adenoma.
Although claim 15 recites confirm that the human subject has the advanced colorectal adenoma, before treating of the human subject for the advanced colorectal adenoma, which is of great generality being routine and conventional as demonstrated in the prior art rejections documented below and is not a particular treatment.
Regarding claim 16, the claim recites: comprising performing a surgical treatment on the human subject.
Although claim 16 recites comprising performing a surgical treatment on the human subject, which is of great generality being routine and conventional as demonstrated in the prior art rejections documented below and is not a particular treatment.
Regarding claim 17, the claim recites: wherein the surgical treatment comprises removing the advanced colorectal adenoma during colonoscopy.
Although claim 17 recites surgical treatment comprises removing the advanced colorectal adenoma during colonoscopy, which is of great generality being routine and conventional as demonstrated in the prior art rejections documented below and is not a particular treatment.
Dependent claims set forth further limitations about the samples, expression levels of miR-15b and miR-29a, housekeeping miRNAs, confirming the presence of disease, treatment after confirmation of disease, and treatment(s).
The instant claim 3 is directed towards: An in vitro method for diagnosing advanced colorectal adenoma in human subject, the method comprising: (a) measuring the expression pattern or level of microRNAs, obtained from a minimally-invasive biological sample of the human subject (b) comparing the expression pattern or level of microRNA, in the biological sample of the human subject with a control expression pattern or level of the microRNAs in a corresponding biological sample of a healthy subject not suffering from advanced colorectal adenoma; and (c) diagnosing the human subject with advanced colorectal adenoma when the microRNAs in the biological sample of the human subject are overexpressed compared to the control expression pattern or level of the microRNAs in the corresponding biological sample of the healthy subject; wherein: the microRNAs comprise at least miR-15b and miR-29a, or at least miR-15b and miR- 29a and miR-18a, or at least miR-15b and miR-29a and miR-19a, or at least miR-15b and miR- 29a and miR-19b, or at least miR-15b and miR-29a and miR-18a and miR-19a and miR-19b; the minimally-invasive biological sample comprises a blood sample, a plasma sample or a serum sample: and the measuring of the expression pattern or levels of the microRNAs is carried using real-time quantitative polymerase chain reaction (PCR) and subject to normalization based on a geometric mean of the expression levels of housekeeping microRNAs including cel- miR-39 and hsa-miR-1228.
The (a) measuring the expression pattern or level … (b) comparing the expression pattern or level … with a control…and (c) diagnosing the human subject with advanced colorectal adenoma when the microRNAs in the biological sample of the human subject are overexpressed compared to the control expression pattern or level of the microRNAs in the corresponding biological sample of the healthy subject” is considered to be an abstract idea relating to organizing or analyzing information in a way that can be performed mentally or is analogous to human mental work and the steps are also considered routine and conventional as demonstrated by the 35 USC § 103 rejections stated below. (See MPEP 2106.04(a)(2) and 2106.05(d)).
The correlation of overexpression of at least miR-15b and miR-29a or the like to advanced colorectal adenomas is a natural phenomenon as the expression is naturally occurring in subjects with advanced colorectal adenoma. (See MPEP 2106.04(b)).
The normalization based on a geometric mean is considered an abstract idea relating to a mathematical concept.
Dependent claims set forth further limitations about confirming the presence of disease, the samples, expression of miR-15b and miR-29a, and housekeeping miRNAs.
The instant claim 4 is directed towards: The method according to claim 3, further comprising: confirming the presence of advanced colorectal adenoma by performing colonoscopic examination on the human subject”.
The confirming the presence of advanced colorectal adenoma is considered an abstract idea relating to organizing or analyzing information in a way that can be performed mentally or is analogous to human mental work.
The performing colonoscopic examination on the human subject is considered routine and conventional as demonstrated by the 35 USC § 103 rejections stated below. (See MPEP 2106.04(a)(2) and 2106.05(d)).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 phenomena.
With regard to claim 1, “A method for treating advanced colorectal adenoma in a human subject, the method comprising: (a) obtaining a biological sample from the human subject; (b) measuring expression levels of at least miR-15b and miR-29a in the biological sample from the human subject; (c) comparing the expression levels of the miR-15b and the miR-29a in the biological sample from the human subject to expression levels of miR-15b and miR-29a in a corresponding biological sample from a healthy subject not suffering from advanced colorectal adenoma; (d) diagnosing the human subject with the advanced colorectal adenoma when both of the miR-15b and the miR-29a in the biological sample from the human subject are overexpressed relative to the expression levels of the miR-15b and the miR- 29a in the corresponding biological sample from the heathy subject; and (e) treating the human subject for the advanced colorectal adenoma by performing a surgical treatment, a chemotherapy treatment, an antibody treatment, an anti-vascular endothelial growth factor receptor (VEGF/R) treatment, a radiation treatment, or any combination thereof, on the human subject, wherein the biological sample comprises a blood sample, a plasma sample or a serum sample.”
The steps (a) obtaining a biological sample … (b) measuring expression levels… (c) comparing the expression levels… in the biological sample from the human subject to expression levels of… in a corresponding biological sample from a healthy subject… (d) diagnosing the human subject with the advanced colorectal adenoma… are an abstract idea relating to organizing or analyzing information in a way that can be performed mentally or is analogous to human mental work and the steps. (See MPEP 2106.04(a)(2)).
The correlation between when both miR-15b and miR-29a in the biological sample from a human subject are overexpressed as compared to the expression levels in a healthy subject to advanced colorectal adenoma is based on a natural phenomenon, as the expression is naturally occurring in subjects with advanced colorectal adenoma. (See 2106.04(b)).
With regard to claim 3, “An in vitro method for diagnosing advanced colorectal adenoma in human subject, the method comprising: (a) measuring the expression pattern or level of microRNAs, obtained from a minimally-invasive biological sample of the human subject (b) comparing the expression pattern or level of microRNA, in the biological sample of the human subject with a control expression pattern or level of the microRNAs in a corresponding biological sample of a healthy subject not suffering from advanced colorectal adenoma; and (c) diagnosing the human subject with advanced colorectal adenoma when the microRNAs in the biological sample of the human subject are overexpressed compared to the control expression pattern or level of the microRNAs in the corresponding biological sample of the healthy subject; wherein: the microRNAs comprise at least miR-15b and miR-29a, or at least miR-15b and miR- 29a and miR-18a, or at least miR-15b and miR-29a and miR-19a, or at least miR-15b and miR- 29a and miR-19b, or at least miR-15b and miR-29a and miR-18a and miR-19a and miR-19b; the minimally-invasive biological sample comprises a blood sample, a plasma sample or a serum sample: and the measuring of the expression pattern or levels of the microRNAs is carried using real-time quantitative polymerase chain reaction (PCR) and subject to normalization based on a geometric mean of the expression levels of housekeeping microRNAs including cel- miR-39 and hsa-miR-1228.”
The (a) measuring the expression pattern or level … (b) comparing the expression pattern or level … with a control…and (c) diagnosing the human subject with advanced colorectal adenoma when the microRNAs in the biological sample of the human subject are overexpressed compared to the control expression pattern or level of the microRNAs in the corresponding biological sample of the healthy subject” is considered to be an abstract idea relating to organizing or analyzing information in a way that can be performed mentally or is analogous to human mental work (See MPEP 2106.04(a)(2)).
The correlation of overexpression of at least miR-15b and miR-29a or the other optional miRNA(s) listed in claim 3 to advanced colorectal adenomas is a natural phenomenon as the expression is naturally occurring in subjects with advanced colorectal adenoma. (See MPEP 2106.04(b)).
The normalization based on a geometric mean is considered an abstract idea relating to a mathematical concept.
With regard to claim 4 “The method according to claim 3, further comprising: confirming the presence of advanced colorectal adenoma by performing colonoscopic examination on the human subject”.
The confirming the presence of advanced colorectal adenoma is considered an abstract idea relating to organizing or analyzing information in a way that can be performed mentally or is analogous to human mental work.
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.
Regarding claim 1 step (d), the claim recites: diagnosing…; and treating the human subject for the advanced colorectal adenoma by performing a surgical treatment, a chemotherapy treatment, an antibody treatment, an anti-vascular endothelial growth factor receptor (VEGF/R) treatment, a radiation treatment, or any combination thereof, on the human subject.
Although claim 1 step (d) recites treating the human subject for the advanced colorectal adenoma by performing a surgical treatment, a chemotherapy treatment, an antibody treatment, an anti-vascular endothelial growth factor receptor (VEGF/R) treatment, a radiation treatment, or any combination thereof, on the human subject, which is of great generality being routine and conventional as demonstrated in the prior art rejections documented below and is not a particular treatment.
Regarding claim 15, the claim recites: further comprising performing colonoscopy on the human subject to confirm that the human subject has the advanced colorectal adenoma, before treating of the human subject for the advanced colorectal adenoma.
Although claim 15 recites confirm that the human subject has the advanced colorectal adenoma, before treating of the human subject for the advanced colorectal adenoma, which is of great generality being routine and conventional as demonstrated in the prior art rejections documented below and is not a particular treatment.
Regarding claim 16, the claim recites: comprising performing a surgical treatment on the human subject.
Although claim 16 recites comprising performing a surgical treatment on the human subject, which is of great generality being routine and conventional as demonstrated in the prior art rejections documented below and is not a particular treatment.
Regarding claim 17, the claim recites: wherein the surgical treatment comprises removing the advanced colorectal adenoma during colonoscopy.
Although claim 17 recites surgical treatment comprises removing the advanced colorectal adenoma during colonoscopy, which is of great generality being routine and conventional as demonstrated in the prior art rejections documented below and is not a particular treatment.
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.
Independent Claim 1 requires the routine and conventional active steps of analysis of a sample based on the overexpression of at least miR-15b and miR-29a in a human subject sample and general treatment, based on the overexpression of at least miR-15b and miR-29a in a human subject sample similar to that of Gironella et al. (“Gironella”; Patent App. Pub. No. AU 2015201072 A1, Mar. 19, 2015, filed on Mar. 3, 2015) as discussed in the 35 U.S.C. 103 rejection below. Thus, the claim does not provide additional steps which are significantly more.
Briefly, Gironella discloses colorectal cancer detection, and more particularly, to plasma microRNAs for the detection of early colorectal cancer. Specifically, the present invention includes methods, kits and biomarkers for diagnosing or detecting colorectal neoplasia in a human subject comprising the steps of: A method for diagnosing or detecting colorectal neoplasia in a human subject comprising the steps of: obtaining one or more biological samples from the subject suspected of suffering from colorectal neoplasia; measuring an overall expression pattern or level of one or more microRNAs obtained from the one or more biological samples of the subject; and comparing the overall expression pattern of the one or more microRNAs from the biological sample of the subject suspected of suffering from colorectal neoplasia with the overall expression pattern of the one or more microRNAs from a biological sample of a normal subject, wherein the normal subject is a healthy subject not suffering from colorectal neoplasia, wherein overexpression of a combination of miR19a and miR19b, or miR19a and miR19b and miR 15b is indicative of colorectal cancer. (Abstract)
Independent Claim 3 requires the routine and conventional active steps of analysis of a sample based on the overexpression of at least miR-15b and miR-29a in a human subject, wherein the measuring of the expression pattern or levels of the microRNAs is carried using real-time quantitative polymerase chain reaction (PCR) and subject to normalization based on a geometric mean of the expression levels of housekeeping microRNAs including cel- miR-39 and hsa-miR-1228 similar to that of Gironella et al. (“Gironella”; Patent App. Pub. No. AU 2015201072 A1, Mar. 19, 2015, filed on Mar. 3, 2015) in view of Hu et al. (“Hu”; (2014). Human miR-1228 as a stable endogenous control for the quantification of circulating microRNAs in cancer patients. International journal of cancer, 135(5), 1187–1194.) and Sun et al. (“Sun”; (2016). Examining plasma microRNA markers for colorectal cancer at different stages. Oncotarget, 7(10), 11434–11449), as discussed in the 35 U.S.C. 103 rejection below. Thus, the claim does not provide additional steps which are significantly more.
Dependent claims of claim 1 and 3 set forth further limitations about the samples, expression levels of miR-15b and miR-29a, housekeeping miRNAs, confirming the presence of disease, and/or and treatment(s) which are all routine and conventional based on Gironella et al. (“Gironella”; Patent App. Pub. No. AU 2015201072 A1, Mar. 19, 2015, filed on Mar. 3, 2015) in view of Hu et al. (“Hu”; (2014). Human miR-1228 as a stable endogenous control for the quantification of circulating microRNAs in cancer patients. International journal of cancer, 135(5), 1187–1194.) and Sun et al. (“Sun”; (2016). Examining plasma microRNA markers for colorectal cancer at different stages. Oncotarget, 7(10), 11434–11449) as discussed in the 35 U.S.C. 103 rejection below.
Response to Arguments
Applicant's arguments filed 01/28/2026 do not apply to the new grounds of rejections necessitated by amendment to the claims. To clarify some instances argued in the response filed 01/28/2026 see responses to each argument made by Applicant below:
Applicants’ argument: “Claim 1 is patent eligible subject matter under 35 U.S.C. § 101, because it integrates the alleged judicial exception into the practical application of treating the subject by performing a surgical treatment, a chemotherapy treatment, an antibody treatment, an anti-vascular endothelial growth factor receptor (VEGF/R) treatment and/or a radiation treatment after the subject is diagnosed advanced colorectal adenoma (AA).”
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 idea/mental steps of comparing, as well as natural correlation of expression levels of miRNA mir-15b and mir- 29a to advanced colorectal adenoma. The newly added claim recites an abstract idea/mental process of comparing, abstract idea/mathematical concept of normalization based on a geometric mean, and a natural correlation of expression pattern/level of miRNA to advanced colorectal adenoma. These judicial exceptions are not integrated into a practical application because the treatment step is of great generality being routine and conventional as demonstrated in the prior art rejections documented below and is not a particular treatment. 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.
Applicants’ argument: “Claim 3 is patent eligible subject matter under 35 U.S.C. § 101, because the claimed method includes additional elements that amount to significantly more than the judicial exception. (Pg. 13) and “Using the geometric mean of the expression levels of cel-miR-39 and hsa-miR-1228 was not well-understood, routine, or conventional at the time that the present application was filed.).”
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. As recited below in the new grounds of 35 U.S.C. 103 rejection on Pg. 20-24, “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 methods of measuring of the expression levels of the at least miR-15b and the miR-29a is carried using real-time quantitative polymerase chain reaction (PCR) and subject to normalization based on levels of housekeeping genes as taught by Gironella to incorporate the method of subjecting the expression levels to normalization based on a geometric mean of the expression levels of housekeeping microRNAs including a C. elegans based external control miRNA and hsa-miR-1228 endogenous control as taught by Hu and incorporate the method of subjecting the expression levels to normalization based the expression levels of housekeeping microRNAs including a cel-miR-1228 external control as taught by Sun and provide a method according to the limitations of claim 3 steps (a-c) and claim 20, wherein the measuring of the expression levels of the at least miR-15b and the miR-29a is carried using real-time quantitative polymerase chain reaction (PCR) and subject to normalization based on a geometric mean of the expression levels of housekeeping microRNAs including cel-miR-39 and hsa-miR-1228. These claim elements were known in the art and one of skill in the art could have combined these elements by known methods with no change in their respective functions, and the combination would have yielded the predictable outcome according to the limitations of claims 3 and 20. Doing so would allow for minimized variability arising from technical, template quality, or experimental efficiency, and ability to control extraction efficiency, reagents in a reaction cocktail, and variations in machine operation parameters (pg. 1192, Col. 2, Para. 2).” Thus, using the geometric mean of the expression levels of cel-miR-39 and hsa-miR-1228 was well-understood, routine, or conventional at the time that the present application was filed.
Claim Rejections - 35 USC § 103
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.
Claim 1, 11-12 and 15-17 are rejected under 35 U.S.C. 103 as being unpatentable over Gironella et al. (“Gironella”; Patent App. Pub. No. AU 2015201072 A1, Mar. 19, 2015, filed on Mar. 3, 2015) as evidenced by Gattolliat et al. (“Gattolliat”; (2015). MicroRNA and targeted mRNA expression profiling analysis in human colorectal adenomas and adenocarcinomas. European journal of cancer (Oxford, England: 1990), 51(3), 409–420.).
Claim interpretations: In the absence of specified physiological, pathological and/or molecular criteria/threshold for a given colorectal cancer/adenoma being classified as “advanced”, the limitation “advanced colorectal adenoma” recited in the preamble of claim 1 encompasses colorectal cancer/adenoma taught by Gironella et al. (2015) and Gattolliat”; (2015) based on expression level of miRNAs.
Gironella discloses colorectal cancer detection, and more particularly, to plasma microRNAs for the detection of early colorectal cancer. Specifically, the present invention includes methods, kits and biomarkers for diagnosing or detecting colorectal neoplasia in a human subject comprising the steps of: A method for diagnosing or detecting colorectal neoplasia in a human subject comprising the steps of: obtaining one or more biological samples from the subject suspected of suffering from colorectal neoplasia; measuring an overall expression pattern or level of one or more microRNAs obtained from the one or more biological samples of the subject; and comparing the overall expression pattern of the one or more microRNAs from the biological sample of the subject suspected of suffering from colorectal neoplasia with the overall expression pattern of the one or more microRNAs from a biological sample of a normal subject, wherein the normal subject is a healthy subject not suffering from colorectal neoplasia, wherein overexpression of a combination of miR19a and miR19b, or miR19a and miR19b and miR 15b is indicative of colorectal cancer. (Abstract)
Regarding claim 1, Gironella teaches a method comprising “human subject … obtaining one or more biological samples from the subject, wherein the biological samples are selected from of one or more biological fluids, a plasma sample, a serum sample, a blood sample” (Pg. 10 ln 19-22.). Thus, Gironella teaches a method comprising step (a) obtaining a biological sample from the human subject; and wherein the biological sample comprises a blood sample, a plasma sample or a serum sample.
Regarding claim 1 steps (b-d), Gironella teaches a method comprising “determining of the level of expression of microRNAs that are overexpressed in colorectal neoplasia are selected from: …hsa-miR-29a; …hsa-miR-15b” (Pg. 32, ln 1-2 and table). Gironella teaches a method comprising “diagnosing or detecting colorectal neoplasia in a human subject comprising the steps of: obtaining one or more biological samples from the subject… measuring an overall expression pattern or level of one or more microRNAs obtained from the one or more biological samples of the subject; and comparing the overall expression pattern of the one or more microRNAs from the biological sample of the subject suspected of suffering from colorectal neoplasia with the overall expression pattern of the one or more microRNAs from a biological sample of a normal subject, wherein the normal subject is a healthy subject not suffering from colorectal neoplasia, wherein overexpression of a combination of miR… and miR15b is indicative of colorectal cancer. In one aspect, the method further comprises the analysis of at least one of … miR29a … as compared to expression from the normal subject is indicative of colorectal neoplasia.” (Pg. 3 ln 22-33). “colorectal neoplasia” reads on advanced adenomas. Thus, Gironella teaches a method comprising steps(b-d: (b) measuring expression levels of at least miR-15b and miR-29a in the biological sample from the human subject; (c) comparing the expression levels of the miR-15b and the miR-29a in the biological sample from the human subject to expression levels of miR-15b and miR-29a in a corresponding biological sample from a healthy subject not suffering from advanced colorectal adenoma; and (d) diagnosing the human subject with the advanced colorectal adenoma when both of the miR-15b and the miR-29a in the biological sample from the human subject are overexpressed relative to the expression levels of the miR-15b and the miR- 29a in the corresponding biological sample from the heathy subject.
Regarding claim 1 step (e), Gironella teaches a method comprising “using … expression signatures for identifying colorectal cancer as well as for preventing or treating such a condition.” (Pg. 2 ln 32 and Pg. 3 ln 1). Gironella teaches a method comprising “treating a patient at risk or suffering from colorectal neoplasia, selecting an anti-neoplastic agent therapy (e.g., nucleic acid crosslinking agents, small molecules, biologics such as monoclonal antibodies with or without cell killing payloads, both targeted and untargeted) for a patient at risk or suffering from colorectal neoplasia” (Pg. 13 ln 5-8). “such a condition” Thus, Gironella teaches a method comprising step (e) treating the human subject for the advanced colorectal adenoma by performing a surgical treatment, a chemotherapy treatment, an antibody treatment, an anti-vascular endothelial growth factor receptor (VEGF/R) treatment, a radiation treatment, or any combination thereof, on the human subject.
Furthermore, Gattolliat discloses “Colorectal cancer (CRC) mainly develops from colorectal adenomas (CRAs). MicroRNAs (miRs) are short non-coding transcripts that regulate gene expression by binding to target mRNAs, preventing their expression. It was suggested that miRs were involved in cancer as tumour suppressors or oncogenes, thereby being also potential cancer biomarkers. We conducted an expression analysis of miRNAs and several of their target mRNAs, by using microarrays and quantitative Reverse Transcription-Polymerase Chain Reaction (RT-PCR) (RT-qPCR), in CRA and CRC, as compared to normal mucosa (NOR), in order to identify candidate miRNAs involved in CRC progression… Conclusions: We confirmed that several miRNAs were abnormally expressed in colorectal lesions, identified new deregulated miRs, and showed that several miRNAs could mark the transition from NOR to CRA, thereby marking progression from the early steps of cancer. (Abstract-Background and Conclusions) “Colorectal adenoma” reads on AA.
Regarding claim 1, Gattolliat teaches upregulation of both of the miR-15b and the miR-29a in biological samples from human subjects with colorectal adenoma compared to a normalsubjects as shown in the highlighted Table 3. (Table 3, shown below). Thus, Gattolliat provides evidence of upregulation of both mir-15b and mir-29a in colorectal adenoma.
PNG
media_image1.png
495
1533
media_image1.png
Greyscale
Regarding claims 11-12, Gironella teaches a method comprising “wherein the biological samples are selected from of one or more biological fluids, a plasma sample, a serum sample, a blood sample” (Pg. 10 ln 19-22). Thus, Gironella teaches a method wherein the biological sample comprises a plasma sample; and wherein the biological sample comprises a serum sample.
Regarding claim 15, Gironella teaches a method comprising “colonoscopy constitutes an invasive approach” (Pg. 18 ln 16-17). Thus, Gironella suggests a method further comprising performing colonoscopy on the human subject to confirm that the human subject has the advanced colorectal adenoma, before treating of the human subject for the advanced colorectal adenoma.
Regarding claims 16-17, Gironella suggest a method comprising “surgery” (Pg. 19 ln 23), “colonoscopy” (Pg. 19 ln 14) and “removal of cancer precursor lesions” (Pg. 18 ln 12). Thus, Gironella suggests a method further comprising performing a surgical treatment on the human subject, wherein the treatment comprises removal of precursor lesion(s) or a colonoscopy.
Therefore, the invention as recited in claims 1, 11-12 and 15-17 is prima facie obvious over the prior art Gironella et al. One of ordinary skill in the art would have had a reasonable expectation of success given the obviousness of the claim limitations over Gironella and as evidenced by Gattolliat, which provides evidence that overexpression of both of the miR-15b and the miR-29a a biological sample from the human subject with colorectal adenoma would be expected (Table 3- shown above) before the filing date of the instant application. These claim elements were known in the art and one of skill in the art could have combined these elements by known methods with no change in their respective functions, and the combination would have yielded the predictable outcome according to the limitations of claims 1, 11-12 and 15-17. Doing so would allow for improved analysis for diagnosis of subjects with advanced colorectal and treatment thereof. It would have been obvious to provide a method for treating advanced colorectal adenomas in a human subject according to the limitations of the instant application claims 1, 11-12 and 15-17 based on Gironella et al. (Patent App. Pub. No. AU 2015201072 A1).
Response to Arguments
Applicant' s arguments filed 01/28/2026 (Pg.15-17) with respect to claim 1, 3-4 and 11-20 have been considered but are do not apply to the new grounds of rejection. As necessitated by claim amendments, the amended claims required a new-grounds of rejection. To clarify some instances argued in the response filed 01/28/2026 see responses to each argument made by Applicant below:
Applicants’ argument: “Applicant respectfully traverses because Gironella does not reasonably suggest comparing the expression levels of miR-l 5b and miR-29a in the context of diagnosing advanced colorectal adenoma (AA), and no reasonable expectation of success would have existed that doing so would have been successful.” (Pg. 15) and “Given that Gironella plainly teaches that ''further studies would be necessary to establish whether there is any miRNA alone or in combination able to detect these pre-malignant lesions accurately," and characterizes the usefulness of plasma miRNAs in patients with AA as an ''interesting research line'' that warrants ''further investigation," it cannot be concluded that this reference reasonable suggests the claimed methods requiring the comparison of expression levels of at least miR-l 5b and miR-29a with any expectation of success.” (Pg. 17).
Response: In response to applicant's arguments against that Gironella does not reasonably suggest comparing the expression levels of miR-15b and miR-29a in the context of diagnosing advanced colorectal adenoma (AA), as stated in the 35 U.S.C. 103 rejection above, “Gironella teaches a method for diagnosing or detecting colorectal neoplasia in a human subject” and “comparing the overall expression pattern of the one or more microRNAs from the biological sample of the subject suspected of suffering from colorectal neoplasia with the overall expression pattern of the one or more microRNAs from a biological sample of a normal subject, wherein the normal subject is a healthy subject not suffering from colorectal neoplasia, wherein overexpression of a combination of miR… and miR15b is indicative of colorectal cancer. In one aspect, the method further comprises the analysis of at least one of … miR29a … as compared to expression from the normal subject is indicative of colorectal neoplasia.” (Pg. 3 ln 22-33). “colorectal neoplasia” reads on advanced adenomas.
Furthermore, as recited in the 35 U.S.C. 103 rejection above, “the invention as recited in claims 1, 11-12 and 15-17 is prima facie obvious over the prior art Gironella et al. One of ordinary skill in the art would have had a reasonable expectation of success given the obviousness of the claim limitations. These claim elements were known in the art and one of skill in the art could have combined these elements by known methods with no change in their respective functions, and the combination would have yielded the predictable outcome according to the limitations of claims 1, 11-12 and 15-17. Doing so would allow for improved analysis for diagnosis of subjects with AA and treatment thereof. It would have been obvious to provide a method for treating advanced colorectal adenomas in a human subject according to the limitations of the instant application claims 1, 11-12 and 15-17 based on Gironella et al. (Patent App. Pub. No. AU 2015201072 A1).”. Additionally, the results would be expected to be variable based on variable options of disease parameters, measurement, controls and analysis allowed in the claimed method, with a reasonable expectation to yield successful results as the limitations of claims 1, 11-12 and 15-17 were known in the art. Lastly, Gattolliat provides evidence that overexpression of both of the miR-15b and the miR-29a a biological sample from the human subject with colorectal adenoma would be expected (Table 3- shown above) before the filing date of the instant application. Thus, Gironella does reasonably suggest comparing the expression levels of miR-15b and miR-29a in the context of diagnosing advanced colorectal adenoma (AA).
Claims 3-4 and 18-20 are rejected under 35 U.S.C. 103 as being unpatentable over Gironella et al. (“Gironella”; Patent App. Pub. No. AU 2015201072 A1, Mar. 19, 2015, filed on Mar. 3, 2015) in view of Hu et al. (“Hu”; (2014). Human miR-1228 as a stable endogenous control for the quantification of circulating microRNAs in cancer patients. International journal of cancer, 135(5), 1187–1194.) and Sun et al. (“Sun”; (2016). Examining plasma microRNA markers for colorectal cancer at different stages. Oncotarget, 7(10), 11434–11449) and as evidenced by Gattolliat et al. ( “Gattolliat”; (2015). MicroRNA and targeted mRNA expression profiling analysis in human colorectal adenomas and adenocarcinomas. European journal of cancer (Oxford, England : 1990), 51(3), 409–420.).
Claim interpretations: In the absence of specified physiological, pathological and/or molecular criteria/threshold for a given colorectal cancer/adenoma being classified as “advanced”, the limitation “advanced colorectal adenoma” recited in the preamble of claim 1 encompasses colorectal cancer/adenoma taught by Gironella et al. (2015) and Gattolliat”; (2015) based on expression level of miRNAs.
Gironella discloses colorectal cancer detection, and more particularly, to plasma microRNAs for the detection of early colorectal cancer. Specifically, the present invention includes methods, kits and biomarkers for diagnosing or detecting colorectal neoplasia in a human subject comprising the steps of: A method for diagnosing or detecting colorectal neoplasia in a human subject comprising the steps of: obtaining one or more biological samples from the subject suspected of suffering from colorectal neoplasia; measuring an overall expression pattern or level of one or more microRNAs obtained from the one or more biological samples of the subject; and comparing the overall expression pattern of the one or more microRNAs from the biological sample of the subject suspected of suffering from colorectal neoplasia with the overall expression pattern of the one or more microRNAs from a biological sample of a normal subject, wherein the normal subject is a healthy subject not suffering from colorectal neoplasia, wherein overexpression of a combination of miR19a and miR19b, or miR19a and miR19b and miR 15b is indicative of colorectal cancer. (Abstract)
Regarding claim 3 steps (a-c), Gironella teaches a method comprising “determining of the level of expression of microRNAs that are overexpressed in colorectal neoplasia are selected from: …hsa-miR-29a; …hsa-miR-15b” (Pg. 32, ln 1-2 and table). Gironella teaches a method comprising “biosignature or assay will include the combination of both over and underexpressed microRNAs, e.g., from those listed hereinabove or disclosed herein.” (Pg. 32, ln 3-4.). Gironella teaches a method comprising “human subject … obtaining one or more biological samples from the subject, wherein the biological samples are selected from of one or more biological fluids, a plasma sample, a serum sample, a blood sample” (Pg. 10 ln 19-22.).
Gironella teaches a method comprising “comparing the overall expression pattern of the one or more microRNAs from the biological sample of the subject suspected of suffering from colorectal neoplasia with the overall expression pattern of the one or more microRNAs from a biological sample of a normal subject, wherein the normal subject is a healthy subject not suffering from colorectal neoplasia, wherein overexpression of a combination of miR… and miRl5b is indicative of colorectal cancer” (Pg. 3 ln 22-31). Gironella teaches a method comprising “diagnosing or detecting colorectal neoplasia in a human subject comprising the steps of: obtaining one or more biological samples from the subject… measuring an overall expression pattern or level of one or more microRNAs obtained from the one or more biological samples of the subject; and comparing the overall expression pattern of the one or more microRNAs from the biological sample of the subject suspected of suffering from colorectal neoplasia with the overall expression pattern of the one or more microRNAs from a biological sample of a normal subject, wherein the normal subject is a healthy subject not suffering from colorectal neoplasia, wherein overexpression of a combination of miR… and miR15b is indicative of colorectal cancer” (Pg. 3 ln 22-31). Gironella teaches a method comprising “the expression level of the one or more microRNAs is measured by… quantitative real-time PCR” (Pg. 13 ln 1-3). Gironella also teaches a method comprising “Several putative housekeeping small nuclear RNAs were analyzed in order to determine the most suitable in our samples (RNU6B, miR16, miR423-5p, RNU48, miR544, miR103, miR525, miR451 )… expression levels of miRNAs were normalized to miRl6 as internal control” (Pg. 21 ln 11-14). “colorectal neoplasia” reads on advanced adenomas. “housekeeping small nuclear RNAs” reads on “miR16” reads on housekeeping miRNA. Thus, Gironella teaches a method comprising the steps (a-c); (a) measuring the expression pattern or level of microRNA obtained from a minimally-invasive biological sample of the human subject (b) comparing the expression pattern or level of microRNA, in the biological sample of the human subject with a control expression pattern or level of the microRNAs in a corresponding biological sample of a healthy subject not suffering from advanced colorectal adenoma; and (c) diagnosing the human subject with advanced colorectal adenoma when the microRNAs in the biological sample of the human subject are overexpressed compared to the control expression pattern or level of the microRNAs in the corresponding biological sample of the healthy subject; and wherein: the microRNAs comprise at least miR-15b and miR-29a, or at least miR-15b and miR- 29a and miR-18a, or at least miR-15b and miR-29a and miR-19a, or at least miR-15b and miR- 29a and miR-19b, or at least miR-15b and miR-29a and miR-18a and miR-19a and miR-19b; the minimally-invasive biological sample comprises a blood sample, a plasma sample or a serum sample; and the measuring of the expression pattern or levels of the microRNAs is carried using real-time quantitative polymerase chain reaction (PCR) and subject to normalization based on a geometric mean of the expression levels of housekeeping microRNAs.
Furthermore, Gironella teaches “further studies would be necessary to establish whether there is any miRNA alone or in combination able to detect these pre-malignant lesions accurately” (Pg. 30, ln 30-31) and “these results indicate that evaluation of the usefulness of plasma microRNAs in patients with AA constitutes an interesting research line and deserves further investigation” (Pg. 31, ln 2-4).
In addition, Gattolliat discloses “Colorectal cancer (CRC) mainly develops from colorectal adenomas (CRAs). MicroRNAs (miRs) are short non-coding transcripts that regulate gene expression by binding to target mRNAs, preventing their expression. It was suggested that miRs were involved in cancer as tumour suppressors or oncogenes, thereby being also potential cancer biomarkers. We conducted an expression analysis of miRNAs and several of their target mRNAs, by using microarrays and quantitative Reverse Transcription-Polymerase Chain Reaction (RT-PCR) (RT-qPCR), in CRA and CRC, as compared to normal mucosa (NOR), in order to identify candidate miRNAs involved in CRC progression… Conclusions: We confirmed that several miRNAs were abnormally expressed in colorectal lesions, identified new deregulated miRs, and showed that several miRNAs could mark the transition from NOR to CRA, thereby marking progression from the early steps of cancer. (Abstract-Background and Conclusions) “Colorectal adenoma” reads on AA.
Regarding claim 1, Gattolliat teaches upregulation of both of the miR-15b and the miR-29a in biological samples from human subjects with colorectal adenoma compared to a normal subjects as shown in the highlighted Table 3. (Table 3, shown below). Thus, Gattolliat provides evidence of upregulation of both mir-15b and mir-29a in colorectal adenoma.
PNG
media_image1.png
495
1533
media_image1.png
Greyscale
However, Gironella et al does not explicitly teach the limitation normalization based on a geometric mean of the expression levels of housekeeping microRNAs including cel-miR-39 and hsa-miR-1228.
Hu discloses Circulating microRNAs are promising biomarkers for non-invasive testing and dynamic monitoring in cancer patients. However, no consensus exists regarding the normalization of circulating microRNAs in the quantification, making the results incomparable. We investigated global circulating microRNA profiles to identify a stable endogenous control for quantifying circulating microRNAs using three cohorts (n = 544), including 168 control individuals (healthy subjects and those with chronic hepatitis B and cirrhosis) and 376 cancer patients (hepatocellular, colorectal, lung, esophageal, gastric, renal, prostate, and breast cancer patients). GeNorm, NormFinder, and coefficient of variability (CV) were used to select the most stable endogenous control, whereas Ingenuity Pathway Analysis (IPA) was adopted to explore its signaling pathways. Seven candidates (miR-1225-3p, miR-1228, miR-30d, miR-939, miR-940, miR-188-5p, and miR-134) from microarray analysis and four commonly used controls (miR-16, miR-223, let-7a, and RNU6B) from literature were subjected to real-time quantitative reverse transcription-polymerase chain reaction validation using independent cohorts. MiR-1228 (CV = 5.4%) with minimum M value and S value presented as the most stable endogenous control across eight cancer types and three controls. IPA showed miR-1228 to be involved extensively in metabolism-related signal pathways and organ morphology, implying that miR-1228 functions as a housekeeping gene. Functional network analysis found that "hematological system development" was on the list of the top networks that associate with miR-1228, implying that miR-1228 plays an important role in the hematological system. The results explained the steady expression of miR-1228 in the blood. In conclusion, miR-1228 is a promising stable endogenous control for quantifying circulating microRNAs in cancer patients. (Abstract)
Regarding claims 3 and 20, Hu teaches a method comprising “gene expression normalization factor for each sample based on the geometric mean of a user-defined number of reference genes” and “miR-1228 was the most stable circulating miRNA in cancer patients and control subjects… miR-1228 should be considered a stable endogenous control for quantifying circulating miRNAs in cancer patients” (Pg. 1190, Statistical analysis, Para. 2). Hu further suggests a method comprising “Generally, external and internal controls are often used for data normalization in miRNA quantification by qRT-PCR. The external control is a spiked-in RNA that is added to a sample before RNA extraction. The levels of target miRNAs will be normalized to the spiked-in control prior to data analysis. Synthetic Caenorhabditis elegans miRNAs are often used as external controls for human samples” (pg. 1192, Col. 2, Para. 2). “external control” reads on Cel-miR-39. Thus, Gironella and Hu suggest a method comprising subject to normalization based on a geometric mean of the expression levels of housekeeping microRNA including hsa-miR-1228 and a C. elegans based external control miRNA.
Sun discloses “Circulating microRNAs (miRNAs) have emerged as promising biomarkers; however, few miRNAs have been reproducible and can be used in clinical practice. In this study, we screened the levels of 754 miRNAs using TaqMan array in 50 individual plasma samples from 10 demographically matched healthy controls and 40 colorectal cancer (CRC) patients (10 each of stage I–IV) and identified 22 miRNAs associated with the presence of and stages of CRC. Then we performed the validation for 11 miRNAs in an independent cohort including 187 CRC cases and 47 healthy controls. Comprehensive analyses showed that plasma miR-96 distinguished stage I–IV CRC from healthy controls with an area under curve (AUC) of 0.740; miR-203 separated stage III–IV CRC patients from stage I–II with an AUC of 0.757; and miR-141 differentiated stage IV CRC from stage I–III patients with an AUC of 0.851. Survival analyses showed that plasma miR-96 and miR-200b were independent prognostic factors for overall survival. Thus, we propose four miRNAs (miR-96, miR-203, miR-141 and miR-200b) as clinically validated circulating biomarkers for CRC prognosis that warrant further evaluation for clinical utility.” (Abstract)
Regarding claims 3 and 20, Sun teaches a method comprising “using quantitative RT-PCR and spiked-in cel-miR-39 as a normalizer” (Pg. 11435, Clinical validation of candidate miRNAs in a second large cohort of CRC patients and controls, Para. 1) and “Expression levels of miRNAs were quantified in duplicate and spiked-in cel-miR-39 was used as normalizers for plasma miRNA quantification” (Pg. 11445, miRNA clinical validation study, Para. 1). cel-miR-39 is interpreted as a C. elegans based external control. Thus, Gironella, Hu and Sun suggest a method comprising subject to normalization based on a geometric mean of the expression levels of housekeeping microRNAs including cel- miR-39 and hsa-miR-1228.
Gironella, Hu and Sun are considered to be analogous to the claimed invention because they are in the same field of evaluating expression levels of serum/plasma miRNAs in the diagnosis of cancer patients. Since, Gironella teaches “further studies would be necessary to establish whether there is any miRNA alone or in combination able to detect these pre-malignant lesions accurately” (Pg. 30, ln 30-31) and “these results indicate that evaluation of the usefulness of plasma microRNAs in patients with AA constitutes an interesting research line and deserves further investigation” (Pg. 31, ln 2-4), an ordinary artisan would be motivated to modify the method suggested by Gironella to improve accuracy of the method for detection of AA. 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 methods of measuring of the expression levels of the at least miR-15b and the miR-29a using real-time quantitative polymerase chain reaction (PCR) and subjecting level to normalization based on levels of housekeeping genes as taught by Gironella and as evidenced by Gattolliat, which provides evidence that overexpression of both of the miR-15b and the miR-29a a biological sample from the human subject with colorectal adenoma would be expected (Table 3- shown above), to incorporate the method of subjecting the expression levels to normalization based on a geometric mean of the expression levels of housekeeping microRNAs including a C. elegans based external control miRNA and hsa-miR-1228 endogenous control as taught by Hu and incorporate the method of subjecting the expression levels to normalization based the expression levels of housekeeping microRNAs including a cel-miR-1228 external control as taught by Sun and provide a method according to the limitations of claim 3 steps (a-c) and claim 20, wherein the measuring of the expression levels of the at least miR-15b and the miR-29a is carried using real-time quantitative polymerase chain reaction (PCR) and subject to normalization based on a geometric mean of the expression levels of housekeeping microRNAs including cel-miR-39 and hsa-miR-1228. These claim elements were known in the art and one of skill in the art could have combined these elements by known methods with no change in their respective functions, and the combination would have yielded the predictable outcome according to the limitations of claims 3 and 20. Doing so would allow for minimized variability arising from technical, template quality, or experimental efficiency, and ability to control extraction efficiency, reagents in a reaction cocktail, and variations in machine operation parameters (pg. 1192, Col. 2, Para. 2).
The teachings of Gironella, Hu and Sun are documented above in the rejection of claims 3 under 35 U.S.C. 103 Claims 4 and 18-20 depend on claim 3.
Regarding claim 4, Gironella teaches a method further comprising “diagnostic colonoscopy” (Pg. 19 ln 14). Thus, Gironella suggests a method comprising confirming the presence of advanced colorectal adenoma by performing colonoscopic examination of the human subject.
Regarding claims 18-19, Gironella teaches a method wherein “the biological samples are selected from of one or more biological fluids, a plasma sample, a serum sample, a blood sample” (Pg. 10 ln 19-22). Thus, Gironella suggests a method wherein the biological sample comprises a plasma sample; and wherein the biological sample comprises a serum sample.
Response to Arguments
Applicant' s arguments filed 01/28/2026 (Pg.15-17) with respect to claims 3-4 and 18-20 have been considered but do not apply to the new grounds of rejection. To clarify some instances argued in the response filed 01/28/2026 please see responses to arguments documented above in the rejection of claims 1, 11-12 and 15-17 under 35 U.S.C. 103.
Claims 1 and 13-14 are rejected under 35 U.S.C. 103 as being unpatentable over Gironella et al. (“Gironella”; Patent App. Pub. No. AU 2015201072 A1, Mar. 19, 2015, filed on Mar. 3, 2015) in view of Hu et al. (“Hu”; (2014). Human miR-1228 as a stable endogenous control for the quantification of circulating microRNAs in cancer patients. International journal of cancer, 135(5), 1187–1194.) and Sun et al. (“Sun”; (2016). Examining plasma microRNA markers for colorectal cancer at different stages. Oncotarget, 7(10), 11434–11449).
The teachings of Gironella are documented above in the rejection of claims 1, 11-12 and 15-17 under 35 U.S.C. 103. Claims 14 depends on claim 13, which depends on claim 1.
Regarding claim 13, Gironella teaches a method comprising “the expression level of the one or more microRNAs is measured by… quantitative real-time PCR” (Pg. 13 ln 1-3). Gironella also teaches a method comprising “Several putative housekeeping small nuclear RNAs were analyzed in order to determine the most suitable in our samples (RNU6B, miR16, miR423-5p, RNU48, miR544, miR103, miR525, miR451 )… expression levels of miRNAs were normalized to miRl6 as internal control” (Pg. 21 ln 11-14). “housekeeping small nuclear RNAs” read on cel-miR-39 and hsa-miR-1228. “internal control” reads on hsa-miR-1228. “miR16” reads on housekeeping miRNA. Thus, Gironella teaches a method wherein the measuring of the expression levels of the at least miR-15b and the miR-29a is carried using real-time quantitative polymerase chain reaction (PCR) and subject to normalization based on levels of housekeeping genes.
Furthermore, Gironella teaches “further studies would be necessary to establish whether there is any miRNA alone or in combination able to detect these pre-malignant lesions accurately” (Pg. 30, ln 30-31) and “these results indicate that evaluation of the usefulness of plasma microRNAs in patients with AA constitutes an interesting research line and deserves further investigation” (Pg. 31, ln 2-4).
However, Gironella does not explicitly teach the limitation normalization based on a geometric mean of the expression levels of housekeeping microRNAs including cel-miR-39 and hsa-miR-1228.
Hu discloses Circulating microRNAs are promising biomarkers for non-invasive testing and dynamic monitoring in cancer patients. However, no consensus exists regarding the normalization of circulating microRNAs in the quantification, making the results incomparable. We investigated global circulating microRNA profiles to identify a stable endogenous control for quantifying circulating microRNAs using three cohorts (n = 544), including 168 control individuals (healthy subjects and those with chronic hepatitis B and cirrhosis) and 376 cancer patients (hepatocellular, colorectal, lung, esophageal, gastric, renal, prostate, and breast cancer patients). GeNorm, NormFinder, and coefficient of variability (CV) were used to select the most stable endogenous control, whereas Ingenuity Pathway Analysis (IPA) was adopted to explore its signaling pathways. Seven candidates (miR-1225-3p, miR-1228, miR-30d, miR-939, miR-940, miR-188-5p, and miR-134) from microarray analysis and four commonly used controls (miR-16, miR-223, let-7a, and RNU6B) from literature were subjected to real-time quantitative reverse transcription-polymerase chain reaction validation using independent cohorts. MiR-1228 (CV = 5.4%) with minimum M value and S value presented as the most stable endogenous control across eight cancer types and three controls. IPA showed miR-1228 to be involved extensively in metabolism-related signal pathways and organ morphology, implying that miR-1228 functions as a housekeeping gene. Functional network analysis found that "hematological system development" was on the list of the top networks that associate with miR-1228, implying that miR-1228 plays an important role in the hematological system. The results explained the steady expression of miR-1228 in the blood. In conclusion, miR-1228 is a promising stable endogenous control for quantifying circulating microRNAs in cancer patients. (Abstract)
Regarding claims 13-14, Hu teaches a method comprising “gene expression normalization factor for each sample based on the geometric mean of a user-defined number of reference genes” and “miR-1228 was the most stable circulating miRNA in cancer patients and control subjects… miR-1228 should be considered a stable endogenous control for quantifying circulating miRNAs in cancer patients” (Pg. 1190, Statistical analysis, Para. 2). Hu further suggests a method comprising “Generally, external and internal controls are often used for data normalization in miRNA quantification by qRT-PCR. The external control is a spiked-in RNA that is added to a sample before RNA extraction. The levels of target miRNAs will be normalized to the spiked-in control prior to data analysis. Synthetic Caenorhabditis elegans miRNAs are often used as external controls for human samples” (pg. 1192, Col. 2, Para. 2). “external control” reads on Cel-miR-39. “hsa-miR-1228” in interpreted as an internal control. Thus, Gironella and Hu suggest a method comprising subject to normalization based on a geometric mean of the expression levels of housekeeping microRNA including hsa-miR-1228 and a C. elegans based external control miRNA.
Sun discloses “Circulating microRNAs (miRNAs) have emerged as promising biomarkers; however, few miRNAs have been reproducible and can be used in clinical practice. In this study, we screened the levels of 754 miRNAs using TaqMan array in 50 individual plasma samples from 10 demographically matched healthy controls and 40 colorectal cancer (CRC) patients (10 each of stage I–IV) and identified 22 miRNAs associated with the presence of and stages of CRC. Then we performed the validation for 11 miRNAs in an independent cohort including 187 CRC cases and 47 healthy controls. Comprehensive analyses showed that plasma miR-96 distinguished stage I–IV CRC from healthy controls with an area under curve (AUC) of 0.740; miR-203 separated stage III–IV CRC patients from stage I–II with an AUC of 0.757; and miR-141 differentiated stage IV CRC from stage I–III patients with an AUC of 0.851. Survival analyses showed that plasma miR-96 and miR-200b were independent prognostic factors for overall survival. Thus, we propose four miRNAs (miR-96, miR-203, miR-141 and miR-200b) as clinically validated circulating biomarkers for CRC prognosis that warrant further evaluation for clinical utility.” (Abstract)
Regarding claims 13-14, Sun teaches a method comprising “using quantitative RT-PCR and spiked-in cel-miR-39 as a normalizer” (Pg. 11435, Clinical validation of candidate miRNAs in a second large cohort of CRC patients and controls, Para. 1) and “Expression levels of miRNAs were quantified in duplicate and spiked-in cel-miR-39 was used as normalizers for plasma miRNA quantification” (Pg. 11445, miRNA clinical validation study, Para. 1). “cel-miR-39” is interpreted as a C. elegans based external control. Thus, Gironella, Hu and Sun suggest a method comprising subject to normalization based on a geometric mean of the expression levels of housekeeping microRNAs including cel- miR-39 and hsa-miR-1228.
Gironella, Hu and Sun are considered to be analogous to the claimed invention because they are in the same field of evaluating expression levels of serum/plasma miRNAs in the diagnosis of cancer patients. Since, Gironella teaches “further studies would be necessary to establish whether there is any miRNA alone or in combination able to detect these pre-malignant lesions accurately” (Pg. 30, ln 30-31) and “these results indicate that evaluation of the usefulness of plasma microRNAs in patients with AA constitutes an interesting research line and deserves further investigation” (Pg. 31, ln 2-4) an ordinary artisan would be motivated to modify the method suggested by Gironella to improve accuracy of the method for detection of AA. 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 methods of measuring of the expression levels of the at least miR-15b and the miR-29a is carried using real-time quantitative polymerase chain reaction (PCR) and subject to normalization based on levels of housekeeping genes as taught by Gironella to incorporate the method of subjecting the expression levels to normalization based on a geometric mean of the expression levels of housekeeping microRNAs including a C. elegans based external control miRNA and hsa-miR-1228 endogenous control as taught by Hu and incorporate the method of subjecting the expression levels to normalization based the expression levels of housekeeping microRNAs including a cel-miR-1228 external control as taught by Sun and provide a method for measuring of the expression levels of the at least miR-15b and the miR-29a is carried using real-time quantitative polymerase chain reaction (PCR) and subject to normalization based on a geometric mean of the expression levels of housekeeping microRNAs including cel-miR-39 and hsa-miR-1228. These claim elements were known in the art and one of skill in the art could have combined these elements by known methods with no change in their respective functions, and the combination would have yielded the predictable outcome according to the limitations of claims 1 and 13-14. Doing so would allow for minimized variability arising from technical, template quality, or experimental efficiency, and ability to control extraction efficiency, reagents in a reaction cocktail, and variations in machine operation parameters (pg. 1192, Col. 2, Para. 2).
Response to Arguments
Applicant' s arguments filed 01/28/2026 (Pg.15-17) with respect to claims 1 and 13-14 have been considered but do not apply to the new grounds of rejection. To clarify some instances argued in the response filed 01/28/2026, please see responses to arguments documented above in the rejection of claims 1, 11-12 and 15-17 under 35 U.S.C. 103.
Conclusion of Response to Arguments
In view of the amendments, new grounds of rejections and above 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.
Any inquiry concerning this communication or earlier communications from the examiner should be directed to KENDRA R VANN-OJUEKAIYE whose telephone number is (571)270-7529. The examiner can normally be reached M-F 9:00 AM- 5:00 PM.
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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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