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 .
Information Disclosure Statement
The IDS received on June 10, 2024 is proper and is being considered by the examiner.
Drawings
The drawings received on January 29, 2024 are acceptable.
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 1-21 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.
Claim 1 is indefinite for reciting the term, “preferably.” When a claim limitation follows the term, “preferably,” the claim becomes indefinite because it is unclear under what conditions the limitation is actively required. The Office notes that there are two instances where the term, “preferably” appears. Both instances should be addressed.
Claims 8, 9, 12, 19, and 20 are indefinite for the same reason.
Claim 1 is also indefinite because the claim is lacking a conjunction between the “using” step and the “detecting” step. For the purpose of prosecution, the conjunction, “and” is assumed.
Claim 12 is also indefinite for the same reason.
Claims 2-11 and 13-21 are indefinite by way of their dependencies on claim 1 and 12, respectively.
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.
Claims 1-21 are rejected under 35 U.S.C. 103 as being unpatentable over Batool et al. (Clinical Cancer Research, published September 15, 2022, vol. 28, pages 4070-4082).
With regard to claim 1, Batool et al. teach a method comprising:
providing a sample comprising a fluid from a subject (“we sought to establish a sensitive plasma-based … assay,” page 4071, 1st column, 1st paragraph);
isolating extracellular vesicles comprising RNA from the sample (“[o]ur extracellular vesicle RNA-based ddPCR”, page 4071, 1st column; also “plasma was obtained from each clinical sample … EV [extracellular vesicle] RNA was eluted …”, page 4071, 2nd column, bottom paragraph);
reverse transcribing the RNA in the presence of 7dG to create cDNA (“EV RNA isolated from plasma samples, was reverse transcribed into cDNA”, page 4072, 1st column; also “First-Strand Synthesis System … protocol was optimized to maximize cDNA yield … 1.0 mL 7-deaza-dGTP … were combined with template RNA”, page 4072, 1st column, Optimized reverse transcription);
using polymerase chain reaction (PCR), amplifying any epidermal growth factor receptor v III (EGFRvIII) sequences present in the cDNA to generate EGFRvIII amplicons (“ddPCR was performed using purified cDNA obtained from reverse transcription of EV RNA isolated from plasma samples”, page 4073, 1st column), wherein the amplicon produced is between 50 and 100 (see “primer sets generated amplicons of varying lengths, ranging from 34 to 428 bp. The key criteria in selecting the optimal primer set included; target amplicon size <100 … further optimized produced a target amplicon of 96bp”, page 4072, 2nd column, Primers and Probes).
With regard to claim 2, the method also involves preamplification (“detection of low levels of mutant EV RNA was enhanced via PCR preamplification”, page 4073, 1st column, 2nd paragraph).
With regard to claims 3-5, the subject is known to have cancer, that is glioblastoma (“plasma derived from EV RNA from patients with glioma”, page 4078, 2nd column, 1st paragraph; “prediction of glioblastoma”, Abstract).
With regard to claims 6 and 7, the primers are identical (see page 4072, 2nd column, Primers and Probes).
With regard to claims 8 and 9, the amplification is performed using ddPCR in betaine (see above, also “ddPCR SuperMix for Probes … 5 mol/L betaine solution”, page 4073, 1st column, bottom paragraph).
With regard to claims 10 and 11, the artisans use the same probe as claim 11 with a detectable moiety FAM/MGBNFQ (see page 4072, 2nd column).
With regard to claim 12, the artisans explicitly teach their method for detecting EGFRvIII positive cancer in a human subject (“we report the potential clinical utility of this technique in patients with glioma with several applications: diagnosis, monitoring tumor progression, and assessing response to therapy,” page 4079, 2nd column).
With regard to claim 14, the method also involves preamplification (“detection of low levels of mutant EV RNA was enhanced via PCR preamplification”, page 4073, 1st column, 2nd paragraph).
With regard to claims 15 and 16, the subject is known to have cancer, that is glioblastoma (“plasma derived from EV RNA from patients with glioma”, page 4078, 2nd column, 1st paragraph; “prediction of glioblastoma”, Abstract).
With regard to claims 17 and 18, the primers are identical (see page 4072, 2nd column, Primers and Probes).
With regard to claims 19 and 20, the amplification is performed using ddPCR in betaine (see above, also “ddPCR SuperMix for Probes … 5 mol/L betaine solution”, page 4073, 1st column, bottom paragraph).
With regard to claim 21, the artisans use the same probe as claim 11 with a detectable moiety FAM/MGBNFQ (see page 4072, 2nd column).
While Batool et al. explicitly suggest that EV are also present in other sources of samples, such as CSF (cerebrospinal fluid, see “liquid biopsy … analysis of nonsolid biological fluid (CSF, plasma), offers new opportunities for GBM molecular characterization and detection …”, page 4070, 1st column), the artisans do not explicitly use CSF in their study (claims 1 and 12, in-part).
While Batool et al. explicitly suggest that their method could be applied to assessing response to therapy (see above), the artisans do not explicitly teach that upon diagnosis, therapy should follow (claim 13).
However, it would have been prima facie obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to apply the knowledge presented by Batool et al. with the purview of the ordinarily skilled artisan to arrive at the invention as claimed for the following reasons.
In KSR, the Supreme Court particularly emphasized “the need for caution in granting a patent based on the combination of elements found in the prior art,” Id. at 415, 82 USPQ2d at 1395, and discussed circumstances in which a patent might be determined to be obvious. Importantly, the Supreme Court reaffirmed principles based on its precedent that “[t]he combination of familiar elements according to known methods is likely to be obvious when it does no more than yield predictable results.” Id. at 415-16, 82 USPQ2d at 1395. The Supreme Court stated that there are “[t]hree cases decided after Graham [that] illustrate this doctrine.” Id. at 416, 82 USPQ2d at 1395. (1) “In United States v. Adams, . . . [t]he Court recognized that when a patent claims a structure already known in the prior art that is altered by the mere substitution of one element for another known in the field, the combination must do more than yield a predictable result.”
As discussed above, Batool et al. already teach that samples such as CSF can be assayed for GBM analysis, and therefore, one of ordinary skill in the art would have had a reasonable expectation of success at applying the method disclosed by Batool et al. to samples already suggested as being useful for such a study, yielding no more than a predictable outcome.
As well, applying conventional means of therapy upon diagnosis of GBM would have been a ntraul conclusion made by the one of ordinary skilled in the art, requiring common sense, said therapy being radiation therapy, as already evidenced by Batool et al. who teach that some of the samples of patients with GBM had been undergoing treatment with radiation therapy (see Table 1 that show chemotherapy, radiotherapy).
Therefore, the invention as claimed is deemed prima facie obvious over Batool et al.
Conclusion
No claims are allowed.
The Office notes that the above cited art (having some common inventors) is the best art.
The art of detecting EGFRvIII via Rt-PCR had been well-established.
For example, Yoshimoto et al. (Clin. Cancer Res., January 2008, vol. 14, no. 2, pages 488-493) teach a method of detecting EGFRvIII mutation in glioblastoma samples via RT-PCR. However, Yoshimoto et al. employ a set of conventional dNTP substrates and do not utilize 7dG during the reverse transcription reaction.
7dG substrates are typically used in a reaction to aid the polymerase when the area to be amplified contains GC-rich regions (see June et al., Mol. Pathol., 2000, vol. 55, pages 55-57):
“CpG islands are GC rich they are prone to form superstructures and display higher melting temperatures. Thus it is difficult to generate PCR products or to obtain a readable sequence from such PCR products. Here we show that the use of 7-deaza-2’-deoxyguanosine (deaza-dGTP) [or 7dG] in PCR reactions allows the generation of full length PCR products …” (Jung et al., at page 55)
, Yoshimoto et al. nor any of the prior art (excepting Batool et al.) teach that the regions amplified when detecting EGFRvIII harbor GC-rich regions or suggest that such a region was difficult to amplify.
Therefore, there would have been no reason to employ 7dG substrates when performing RT-PCR of EGFRvIII.
This determination is consistent with the discovery statement made by Applicants:
“The present inventors observed that the EGFRvIII mRNA transcript contains several four guanine (4G) repeat sequences, especially close the junction site (exon1:exon8), which is also the target region of interest. In other sequences, 4G sequences have been shown to play a role in formation of G-quadruplexes as well as other 3-D secondary structures that are known to play an inhibitory role in reverse transcription and PCR amplification. addition of PCR additives may destabilize secondary structures, as previously reported for the amplification of the highly GC rich TERT promoter region (page 10)
“To the best of the present inventor’s knowledge, RNA secondary structures in EGFRvIII mRNA have not been previously discussed in the literature. Here we sought to establish a sensitive assay for the detection of the EGFRvIII mutation in EV-derived RNA, optionally using plasma-based droplet digital PCR (ddPCR). As described herein, several variables underwent optimization, including minimizing the adverse effects of secondary structures at the reverse transcription and PCR steps, by using different PCR additives. Further, to improve amplification efficiency and specifically we optimized thermocycling conditions, temperature gradients, and purification protocols. Using the overall optimized protocol, the prevalence of EGFRvIII mRNA was determined in tumor tissue from 37 tumor tissue samples. (page 10)
As well, claims satisfy the patent eligibility requirement under 35 U.S.C. 101 because the judicial exception that exists between EGFRvIII mutation in a sample and its correlation to cancer is significantly applied by the use of 7dG during the amplification reaction, (i.e., adding significantly more to the judicial exception).
Inquiries
Any inquiry concerning this communication or earlier communications from the Examiner should be directed to Young J. Kim whose telephone number is (571) 272-0785. The Examiner can best be reached from 7:30 a.m. to 4:00 p.m (M-F). The Examiner can also be reached via e-mail to Young.Kim@uspto.gov. However, the office cannot guarantee security through the e-mail system nor should official papers be transmitted through this route.
If attempts to reach the Examiner by telephone are unsuccessful, the Examiner's supervisor, Gary Benzion, can be reached at (571) 272-0782.
Papers related to this application may be submitted to Art Unit 1681 by facsimile transmission. The faxing of such papers must conform with the notice published in the Official Gazette, 1156 OG 61 (November 16, 1993) and 1157 OG 94 (December 28, 1993) (see 37 CFR 1.6(d)). NOTE: If applicant does submit a paper by FAX, the original copy should be retained by applicant or applicant’s representative. NO DUPLICATE COPIES SHOULD BE SUBMITTED, so as to avoid the processing of duplicate papers in the Office. All official documents must be sent to the Official Tech Center Fax number: (571) 273-8300. Any inquiry of a general nature or relating to the status of this application should be directed to the Group receptionist whose telephone number is (571) 272-1600.
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/YOUNG J KIM/Primary Examiner
Art Unit 1637 February 9, 2026
/YJK/
Glioblastoma (GBM) is the most common and aggressive primary brain tumor with a global incidence of 0.5 to 5 per 100,000 persons and a median survival time of 15 months from the time of diagnosis. Despite increasing advances in therapeutics, prognosis remains poor with high rates of treatment failure and relapse. (page 1)
Diagram depicting nucleotide sequence of the target amplicon … in the deleted variant of the EGFR transcript (EGFRvIII) (top, SEQ ID NO: 1) and wild type EGFR (bottom … SEQ ID NO: 2) (page 4)
Tissue biopsy is the current gold standard diagnostic tool for GBM. However, in addition to being invasive and not always feasible, tissue biopsy only provides a single time point evaluation of the tumor genomic profile, thereby limiting its utility in exploring the tumor heterogeneity spatially and over time. Therefore, there remains the need for development and clinical implementation of alternative sensitive, non-invasive and modalities to facilitate serial sampling and real-time monitoring of evolving genetic and epigenetic alterations. In this regard, liquid biopsy, defined as sampling and analysis of non-solid biological fluids (CSF, plasma), offers new opportunities for GBM molecular characterization and detection prognostic markers. Tumors including gliomas have been shown to shed biological material into the circulation which in turn reflects the transcriptomic and proteomic profile of cell of origin. Examples of analytes include extracellular vesicles (EVs), circulating tumor cells (CTCs), cell-free DNA (cfDNA), protein and metabolites. Tumor derived extracellular vesicles (EVs) are particularly promising the biomarker discovery because they encapsulate and retain tumor specific mRNAs in a stable form in a range of biofluids. In addition, the enclosed cargo can vary in response to diverse stimuli and tumor stage (page 9)
Previous work established detection of several molecular markers in biofluids from patients with gliomas, including EGFRvIII mRNA, IDH1 RNA/DNA, TERT promoter, and PTEN deletion. Epidermal growth factor receptor (EGFR) amplification represents one of the hallmark alterations of GBM and is present in about 57% of primary GBMs and 8% of GBMs arising from recurrent low grade gliomas. Approximately, 30-50% of EGFR-amplified GBM harbor an in-frame deletion of exons 2-7, forming a truncated receptor with loss of extracellular ligand binding domain. This deleted EGFR variant III (EGFRvIII) is capable of constitutive signaling in a ligand independent manner thereby promoting tumorigenesis and overall tumor growth. In contrast to EGFR wild type (EGFRwt), EGFRvIII is only expressed in tumor cells, thereby making it a viable therapeutic and diagnostic target in precision oncology (page 9-10)
The present inventors observed that the EGFRvIII mRNA transcript contains several four guanine (4G) repeat sequences, especially close the junction site (exon1:exon8), which is also the target region of interest. In other sequences, 4G sequences have been shown to play a role in formation of G-quadruplexes as well as other 3-D secondary structures that are known to play an inhibitory role in reverse transcription and PCR amplification. addition of PCR additives may destabilize secondary structures, as previously reported for the amplification of the highly GC rich TERT promoter region (page 10)
To the best of the present inventor’s knowledge, RNA secondary structures in EGFRvIII mRNA have not been previously discussed in the literature. Here we sought to establish a sensitive assay for the detection of the EGFRvIII mutation in EV-derived RNA, optionally using plasma-based droplet digital PCR (ddPCR). As described herein, several variables underwent optimization, including minimizing the adverse effects of secondary structures at the reverse transcription and PCR steps, by using different PCR additives. Further, to improve amplification efficiency and specifically we optimized thermocycling conditions, temperature gradients, and purification protocols. Using the overall optimized protocol, the prevalence of EGFRvIII mRNA was determined in tumor tissue from 37 tumor tissue samples. (page 10)
The assay revealed a higher prevalence of EGFRvIII in EGFR amplified tumor tissues (~80%) as compared to what was previously reported in the literature. (page 10). Using this platform, we showed clinical usefulness of this technique in patients with glioma with several applications: diagnosis, monitoring tumor progression, and assessing response to therapy. (page 10-11)
There are two main types of algorithms commonly used for RNA secondary structure prediction: Deterministic Dynamic programming algorithm, and Minimum free energy algorithm. The former is based mainly on the measurement of frequency of base pair interactions with assumption that the formed base pairs are discontinuous, thereby having low accuracy. Based on the Nussinov algorithm, Zuker proposed a minimum free energy algorithm, a more robust prediction tool based on the principle of interaction of secondary structures with free energy and neighboring base pairs. Hence, this was also used for predicting secondary structures in EGFRvIII RNA. Structural analysis of the predicted 3D foldings using crystallography and other sophisticated modalities is beyond the scope of this study. However, it is worth mentioning that thermodynamically stable secondary structures were also found to be highly prevalent close to the junction site (fusion of exon 1 and exon 8), a nucleotide sequence unique to EGFRvIII mutation. This presented a challenge in primer design and selection due to primer dimers and Tm mismatch. This could potentially lead to non-specific products thereby limiting the specificity of the assay. In addition, secondary structures have a well-documented role in reducing the efficiency of polymerase activity and overall efficacy of PCR amplification. Hence, we sought to overcome these challenges through reverse transcription enhancement via 7-dG and development of an amplification protocol that effectively utilized betaine as a potent reaction buffer. 7-dG, a modified nucleotide, is capable of selectively blocking Hoogsten bond formation, without interfering with normal Watson-Crick base pair interactions. A role for betaine in improving amplification of GC rich regions in TERT promoter mutation has also been reported previously where it was used in conjunction with EDTA. However, while we did see an improvement in separation with EDTA, there was a simultaneous lower mutant fluorescent signal. Hence, we finally used betaine alone at optimized concentration of 0.5 mM. based on the present findings of the prevalence of secondary structures in EGFRvIII, future studies are necessary to further elucidate EGFRvIII structural biology. Additional optimization measures included amplicon length and ddPCR optimization. Short amplicon length, optimized for fragmented plasma nucleic acid combined with betaine, known to act as an iso-stabilizing agent, significantly improved the binding of oligonucleotides to the target region (page 10-11)
Using the optimized assay, we were also able to detect EGFRvIII mutation with a higher sensitivity in tumor tissues. (page 12)