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 .
Status of the Claims
Claims 1-17 and 19-21 are pending and examined herein.
Priority
This application, filed 12/29/2023, is a 371 of PCT/US2022/035342, filed 06/28/2022, which claims benefit of U.S. Provisional Patent Application 63/216,362, filed 06/29/2021. The benefit is acknowledged and the claims examined herein are treated as having an effective filing date of 06/29/2021.
Claim Objections
Claim 20 is objected to because of the following informalities: Claim 20 recites “phorsphorylated glucost transporter 1,” which appears to contain typographical errors. It should be amended for clarity, for example, to recite “phosphorylated glucose transporter type 1.” Appropriate correction is required.
Claim Rejections - 35 USC § 112
The following is a quotation of the first paragraph of 35 U.S.C. 112(a):
(a) IN GENERAL.—The specification shall contain a written description of the invention, and of the manner and process of making and using it, in such full, clear, concise, and exact terms as to enable any person skilled in the art to which it pertains, or with which it is most nearly connected, to make and use the same, and shall set forth the best mode contemplated by the inventor or joint inventor of carrying out the invention.
The following is a quotation of the first paragraph of pre-AIA 35 U.S.C. 112:
The specification shall contain a written description of the invention, and of the manner and process of making and using it, in such full, clear, concise, and exact terms as to enable any person skilled in the art to which it pertains, or with which it is most nearly connected, to make and use the same, and shall set forth the best mode contemplated by the inventor of carrying out his invention.
Claims 5-7, 11-13, 15-17, are rejected under 35 U.S.C. 112(a) or 35 U.S.C. 112 (pre-AIA ), first paragraph, because the specification, while being enabling for certain limited embodiments involving immunostaining/imaging of DCIS breast tissue samples and computer-assisted analysis of intracellular localization patterns of PFKL, PFKFB4, and/or phospho-GLUT1 in those DCIS samples, does not reasonably provide enablement for predicting cancer recurrence, distinguishing recurrent from non-recurrent cancer, or treating a subject predicted to have cancer recurrence across the full scope of the claims. The specification does not enable any person skilled in the art to which it pertains, or with which it is most nearly connected, to use the claimed invention commensurate in scope with these claims.
The enablement requirement is such that the specification of the described invention in such terms that one skilled in the art can make and use the invention to ensure that the invention is communicated to the interested public in a meaningful way. (MPEP 2164)
The standard for determining whether the specification meets the enablement requirement is determined in view of the Wands factors (MPEP 2164.01(a)), to assess whether any necessary experimentation required by the specification is "reasonable" or is "undue." The factors to be considered in determining whether undue experimentation is required include: (1) the quantity of experimentation needed to make or use the invention based on the content of the disclosure, (2) the amount of direction provided by the inventor, (3) the existence of working examples, (4) the nature of the invention, (5) the state of the prior art, (6) the level of one of ordinary skill, (7) the level of predictability in the art, and (8) the breadth of the claims. While all of these factors are considered a sufficient amount for a prima facie case are discussed below.
The breadth of the claims
The claims are substantially broader than the working disclosure. Claim 5 recites that “the subject has cancer or has previously had cancer” and that “peripheral intracellular localization of PFKL and/or PFKFB4 predicts cancer recurrence.” Claim 6 recites that “peripheral intra-cellular localization of PFKL, PFKFB4, and/or pGLUT1 predicts cancer recurrence.” Claim 7 recites that “peripheral intra-cellular localization of PFKL, PFKFB4, and/or the one or more additional biomarkers predicts cancer recurrence.” Claim 11 further expands the cancer to “breast cancer, prostate cancer, lung cancer, melanoma, kidney cancer, thyroid cancer, pancreatic cancer, stomach cancer or bladder cancer.” Claim 12 further recites “ductal carcinoma in situ of the breast, lobular carcinoma in situ, atypical ductal hyperplasia, or atypical lobular hyperplasia.” Claim 13 recites “ipsilateral breast cancer recurrence.” Claim 20 broadly recites “distinguishing recurrent from non-recurrent cancer” by determining intracellular localization of a biomarker for cancer recurrence. Claim 21 recites that “peripheral intracellular localization of the biomarker for cancer recurrence indicates recurrent cancer.”
The claims therefore cover multiple cancer types, multiple breast disease subtypes, multiple biomarkers, multiple recurrence determinations, and broad clinical use of localization patterns to predict or indicate recurrence. Claims 15-17 and 19 are broader still because they add treatment steps. Claim 15 recites “treating a subject predicted to have cancer recurrence with surgery.” Claim 16 recites “treating a subject predicted to have cancer recurrence with administration of inhibitors to enzyme or transporter accumulation at plasma membrane.” Claim 17 recites that the inhibitors comprise “colchicine, taxol, a calmodulin antagonist, a prenylation inhibitor, an anesthetic, or combinations thereof.” Claim 19 recites a “treatment regimen” comprising “surgery; administration of an inhibitor to enzyme or transporter accumulation at plasma membrane; immunotherapy; radiotherapy; and administration of a chemotherapeutic agent.”
The claims do not require that the sample be a DCIS sample. The claims do not require the specific machine-learning approach disclosed in the specification. The claims do not require the particular imaging method used in the examples. The claims do not require a particular threshold, classifier, recurrence endpoint, patient cohort, training dataset, validation dataset, staining intensity scale, image-acquisition protocol, or localization-scoring method. The claims also do not require any specific treatment-selection rule, dosage, route, timing, treatment duration, or clinical response endpoint for the treatment claims.
Thus, the claims cover substantially more than the specification teaches and substantially more than the limited embodiments for which the specification provides working examples.
The nature of the invention
The invention is not merely directed to detecting PFKL, PFKFB4, or pGLUT1 in a biological sample. The claims require clinically meaningful use of intracellular localization to predict cancer recurrence, distinguish recurrent from non-recurrent cancer, and treat a subject predicted to have recurrence.
The amount of direction provided by the inventor / The existence of working examples
The amount of direction and the working examples are limited. The specification gives examples primarily involving DCIS samples, breast tissue sections, immunostaining, fluorescence microscopy, computer-assisted classification, and a limited recurrence/non-recurrence dataset.
The figure descriptions show this narrow focus. Figure 2 presents representative intracellular patterns of PFKL and PFKFB4 within tissue sections from surgical biopsies of DCIS patients subsequently found to exhibit non-recurrent or recurrent cancer. In the figure, DCIS tissue samples from patients who did not experience a cancer recurrence (A, B) and did experience a cancer recurrence (C, D) are shown. Both PFKL (A) and PFKFB4 (B) were found in a central distribution within epithelial cells of patients who did not experience a recurrence. Panels C and D show PFKL and PFKFB4 labeling of a sample from a patient who subsequently experienced recurrent cancer. These enzymes often adopt a peripheral pattern in epithelial cells of biopsies of patients who later experienced a recurrence (p. 3). These disclosures are directed to DCIS tissue samples and selected representative images. They do not provide working examples for prostate cancer, lung cancer, melanoma, kidney cancer, thyroid cancer, pancreatic cancer, stomach cancer, or bladder cancer as recited in claim 11.
Figure 3 indicates that flux-controlling glycolytic steps may relocate to the apical surface of ductal epithelial cells in DCIS. This figure shows the apical accumulation of PFKL, PFKFB4, and phospho-GLUT1 biomarkers in a subpopulation of DCIS cases that exhibited recurrences. The figure discloses that two of 50 recurrent patient samples exhibited extensive accumulation of three glycolytic metabolon biomarkers at the apical surface of pre-invasive epithelial cells (p. 4). This evidence is particularly important because the specification itself characterizes the phospho-GLUT1/PFKL/PFKFB4 apical accumulation as occurring in “a subpopulation of DCIS cases” and in “two of 50 recurrent patient samples.” This does not reasonably enable the full scope of claim 6 or claim 7, which broadly recite that peripheral intracellular localization of PFKL, PFKFB4, and/or pGLUT1, or PFKL, PFKFB4, and/or one or more additional biomarkers, predicts cancer recurrence.
Figure 4 presents computer findings and that Panel A shows cross-validation studies of machine-based predictions of breast cancer recurrences. The precision and recall curves of a computer training dataset created using micrographs of DCIS samples from patients exhibiting cancer recurrences and those not exhibiting a recurrence are shown. Also, Figure 4 highlights that machine-based classifications of DCIS micrographs were obtained for phospho-GLUT1, PFKL, and PFKFB4-labeled DCIS sections (p. 5). This shows that the disclosed predictive performance depends on a specific computer training dataset created using DCIS micrographs. The claims do not require the computer training dataset, the DCIS micrographs, the disclosed classifier, or the specific labeling conditions. The specification therefore does not teach how to make a recurrence prediction or recurrent/non-recurrent cancer distinction across the full claim scope without developing additional cancer-specific datasets and classifiers.
Figure 5 depicts the minimum number of micrographs required for correct recurrence prediction. It states that twenty-nine consecutive samples from patients subsequently reporting a recurrence were studied. The sections were stained with anti-phospho-GLUT1 then imaged. Each image was examined in acquisition order by computer to assess each micrograph's predicted outcome (p. 5). Also, as more micrographs were recorded for each patient, the number of correct recurrence predictions increased. Based upon these data, at least 10 images per patient were analyzed. (In the case of non-recurrent patients, all images must score as non-recurrent.) The curve does not reach a fraction of 1.0 because 1 patient recurrence was computationally predicted by the PFKL staining patterns, and did not demonstrate a recurrence prediction for the phospho- GLUT1 biomarker (p. 5). This evidence confirms that the disclosed prediction was not a simple visual determination applicable across the full scope. Rather, the disclosed embodiment required multiple micrographs, staining, imaging, and computer assessment. The claims, however, are not limited to this implementation.
Also, the detailed description is likewise broad and conclusory outside the DCIS examples. Specifically, it states that the present disclosure provides methods of predicting cancer recurrence in a subject, methods of preventing cancer recurrence in a subject and methods for distinguishing recurrent from non-recurrent cancer (p. 12). Also, the description discloses that the methods comprise determining intracellular localization of at least one biomarker (e.g., PFKL, PKKFB4, pGLUT1, etc.) for cancer recurrence in a sample from a subject comprising cancer cells, and in some embodiments, peripheral intracellular localization of at least one biomarker (e.g., PFKL, PKKFB4, pGLUT1, etc.) predicts cancer recurrence or indicates recurrent cancer (p. 12). These statements recite the desired result but do not provide enabling guidance across the full claim scope beyond the narrower DCIS examples described in the specification. Furthermore, the specification defines “peripheral intracellular localization” as when localization is not centrally or homogeneously located throughout the cell but rather the localization is towards the edges of the cell near the cell membrane (p.12). This definition explains the meaning of the localization term, but it does not teach how to reliably use that localization to predict recurrence across multiple cancers, multiple breast subtypes, and multiple biomarkers.
The specification further disclose that the cancer may be a carcinoma, sarcoma, lymphoma, leukemia, melanoma, mesothelioma, multiple myeloma, or seminoma. Also, the cancer may be a cancer of the bladder, blood, bone, brain, breast, cervix, colon/rectum, endometrium, head and neck, kidney, liver, lung, muscle tissue, ovary, pancreas, prostate, skin, spleen, stomach, testicle, thyroid or uterus. Further, in some embodiments, the cancer is selected from breast cancer, prostate cancer, lung cancer, melanoma, kidney cancer, thyroid cancer, pancreatic cancer, stomach cancer or bladder cancer (p. 13). Here, the specification does not provide recurrence-prediction examples for each listed cancer. The specification does not teach that PFKL, PFKFB4, pGLUT1, or the additional biomarkers have the same localization-recognition rule, recurrence association, sensitivity, specificity, precision, recall, or clinical utility across these cancers. The specification further indicates that the breast cancer may comprise ductal carcinoma in situ of the breast (DICS), lobular carcinoma in situ (LCIS), atypical ductal hyperplasia (ADH), or atypical lobular hyperplasia (ALH) (p. 13). However, the specification does not provide working examples showing that peripheral localization predicts recurrence in LCIS, ADH, or ALH. The specification also does not provide working examples specifically enabling “ipsilateral breast cancer recurrence” as recited in claim 13.
The experimental section confirm the narrow factual basis, stating that DCIS samples were studied from 101 women (51 non- recurrent, 50 recurrent) who were followed for at least 10 years, and tissue samples were from partial or total mastectomies of women aged 37—80 years after informed consent was obtained (p. 22). These disclosures show that the data are DCIS-specific and do not represent the full cancer genus recited in claim 11. Furthermore, fluorescence microscopy was performed using a Nikon TE2000-U inverted microscope. Images were captured and processed with Metamorph software, and to reduce shot noise, each micrograph was an average of 10-15 images, with each image acquired for 0.2 sec (p. 24). Then, micrographs were evaluated and auto-scaled using ImageJ software. Here, this provides procedural guidance for imaging, but does not provide clinical validation or recurrence-prediction rules across the claimed cancer types. Moreover, the specification discloses that using DCIS lesions of all morphologies, the PFKL and PFKFB4 computer outcome predictions were correct in 92% of the cases, with 2% false positives and 5% false negatives, and using data concerning phospho-GLUT1 accumulation in the plasma membrane’s vicinity for all DCIS morphologies, some of which were recently reported, it was found that the outcomes were correct in 80% of the cases, with 11% false positives and 9% false negatives. The machine test correctly identified 88.1% of the cases, with 11.9% false positives and 0% false negatives for our study population (Fig. 4B) (p. 29). The specification discloses that this is an optimal experimental finding because false negatives must be avoided for clinical use (p. 29). However, these results are limited to the disclosed DCIS image-classification dataset. They do not show enablement of claims 5-7, 11-13, or 20-21 across the broad cancer genus and biomarker combinations recited.
Lastly, the treatment claims are especially unsupported by enabling guidance. Specifically, the specification discloses that the methods may further comprise treating a subject predicted to have cancer recurrence. The treatment or therapeutic regimen may include, but is not limited to, surgery, administration of an inhibitor of enzyme accumulation at the plasma membrane immunotherapy, radiotherapy, administration of a chemotherapeutic agent. Also, it states that in some embodiments, the treatment or therapeutic regimen comprises administration of inhibitors to enzyme or transporter accumulation at plasma membrane. Inhibitors to enzyme accumulation at plasma membrane include, but are not limited to, colchicine, taxol, calmodulin antagonists, anesthetics and prenylation inhibitors (p. 14). These passages provide lists of possible treatment categories and compounds, but they do not teach how to select a treatment based on the localization result. They do not teach the dose, route, schedule, duration, treatment threshold, patient selection criteria, cancer-specific treatment protocol, recurrence-risk cutoff, expected efficacy, or safety considerations. Therefore, the treatment claims require undue experimentation to practice across their full scope.
The state of the prior art / the level of predictability in the art
The state of the prior art and the level of predictability weigh against enablement because the art shows that PFKFB4 and PFKL localization/expression are cancer-context-dependent and require clinical validation. Yao et al. (High Expression of Metabolic Enzyme PFKFB4 Is Associated with Poor Prognosis of Operable Breast Cancer. Cancer Cell International. Vol. 19. No. 1, June 2019) demonstrates that PFKFB4 prognostic work in breast cancer was limited and required validation. Yao states, “PFKFB4 expression was evaluated by immunohistochemistry in surgical specimens retrospectively collected from 200 patients with histologically proven invasive ductal breast cancer” (Abstract, p. 1). Yao further states, “Kaplan–Meier survival analysis and Cox regression analysis were performed to assess the prognostic significance of PFKFB4 expression” (Abstract, p. 1). Yao further states, “This study has some limitations” (p. 6). Specifically, Yao states, “First, this is a retrospective study, in which we tested the association between PFKFB4 expression with DFS and OS in breast cancer rather than true prediction” (p. 6). Yao further states, “Additional study is needed to validate the prognostic value of the novel marker” (p. 6). Yao further states, “Larger cohorts are therefore required before PFKFB4 can be recommended for clinical practice” (p. 6).
Thus, even for PFKFB4 expression in a 200-patient invasive ductal breast cancer cohort, Yao expressly states that the study was “retrospective,” was “rather than true prediction,” and required “Additional study” and “Larger cohorts” before clinical practice. This supports lack of enablement across the full scope of the present claims because the present specification attempts to claim broad cancer recurrence prediction and treatment based on intracellular localization without providing comparable validation across the full scope.
Yun et al. (PFKFB4 as a Prognostic Marker in Non-Muscle-Invasive Bladder Cancer. Urologic Oncology. Vol. 30, No. 6, November 2012 - IDS dated 01/09/2026) similarly demonstrates that PFKFB4 prognostic use in bladder cancer was not predictably enabled without validation. Yun states, “One hundred ninety-three primary NMIBC tissue specimens were analyzed by real-time PCR” (Abstract, p. 894). Yun states, “Immunohistochemical staining was performed on 77 subsets of tumor samples” (Abstract, p. 894). Yun further states, “PFKFB4 localized primarily to the cytoplasm” (p. 895). Yun further states, “Multivariate survival analysis demonstrated that the level of PFKFB4 expression was not an independent predictor of tumor recurrence” (p. 896). Yun further states, “There are several limitations to the current study” (p. 898). Specifically, Yun states that “First, conventional multivariable analyses such as our study are not sufficient to demonstrate improved prediction of outcomes” (p. 898). Yun further states, “Predictive models, including or excluding any new putative biomarker, need to show clinically significant improvement of performance in order to claim any real benefit” (p. 898.) Yun further states, “For overcoming this issue, external validation is crucial” (p. 898).
Yun is particularly relevant to claim 11 because claim 11 expressly covers bladder cancer. Yun shows that even in bladder cancer, PFKFB4 was not a simple recurrence predictor. Yun states that PFKFB4 expression “was not an independent predictor of tumor recurrence” and that “external validation is crucial.” Therefore, the present specification’s DCIS-focused disclosure does not enable broad prediction of recurrence or recurrent/non-recurrent classification in bladder cancer without undue experimentation across the full scope of claim 11.
Kohnhorst et al. (Identification of a Multienzyme Complex for Glucose Metabolism in Living Cells. Journal of Biological Chemistry. Vol. 292, No. 22, June 2017 - IDS dated 01/09/2026) demonstrates that PFKL intracellular localization is mechanistically complex and context-dependent. Kohnhorst states, “human liver-type phosphofructokinase 1 (PFKL), which catalyzes a bottleneck step of glycolysis, forms various sizes of cytoplasmic clusters in human cancer cells” (Abstract, p. 9191). Kohnhorst further states, “these PFKL clusters colocalize with other rate-limiting enzymes in both glycolysis and gluconeogenesis, supporting the formation of multienzyme complexes” (Abstract, p. 9191). Kohnhorst further states, “the direction of glucose flux between glycolysis, the pentose phosphate pathway, and serine biosynthesis seems to be spatially regulated by the multienzyme complexes in a cluster-size-dependent manner” (Abstract, p. 9191).
Kohnhorst further states, “PFKL-mEGFP displays three different sizes of cytoplasmic fluorescent clusters in transfected HeLa and Hs578T cells” (Figure 1, p. 9193). Kohnhorst further states, “Large-sized clusters are found in various cancer cells, but not in non-cancerous human breast tissue cells (Hs578Bst)” (p. 9192). Kohnhorst further states, “we conclude that neither the formation of the fluorescent clusters by PFKL-mEGFP, nor their size, is governed by the expression levels of PFKL-mEGFP at single-cell levels” (p. 9194). Kohnhorst further states, “the formation of medium- and large-sized clusters at single-cell levels corresponds to the metabolic shunts of glucose flux into the pentose phosphate pathway and serine biosynthesis, respectively” (p. 9198). Kohnhorst further states, “although extensive functional studies are required” (p. 9199).
Kohnhorst therefore shows that intracellular localization of PFKL is not a simple binary feature that can be broadly applied to all cancers as a recurrence predictor. Instead, PFKL localization involves cluster size, colocalization with other enzymes, glucose flux, acetylation, cancer cell context, and functional metabolic state. This supports the conclusion that a PHOSITA would require substantial experimentation to apply peripheral localization of PFKL/PFKFB4/pGLUT1 to predict recurrence across the full scope of the claims.
The level of ordinary skill in the art
The level of ordinary skill in the art would have been high. A PHOSITA would likely have had advanced training (e.g., Ph.D. or M.D.) and experience in molecular pathology, cancer biology, immunostaining, fluorescence microscopy, biomarker validation, image analysis, statistical modeling, and clinical oncology. However, this high level of skill does not compensate for the absence of enabling disclosure commensurate with the full claim scope. Accordingly, even a skilled artisan would need to perform extensive experimentation to practice the full scope of the claims, including developing cancer-specific staining protocols, collecting recurrent and non-recurrent cohorts, training and validating classifiers, determining localization thresholds, correlating localization with recurrence endpoints, and developing treatment-selection protocols.
The quantity of experimentation needed to make or use the invention based on the content of the disclosure
The quantity of experimentation required to practice the full scope of claims 5-7, 11-13, 15-17, 19, and 20-21 would be extensive and undue. For claims 5-7 and 20-21, a PHOSITA would need to determine whether peripheral intracellular localization of PFKL, PFKFB4, pGLUT1, and/or the additional biomarkers predicts cancer recurrence or indicates recurrent cancer for each claimed cancer type. This would require identifying appropriate samples, establishing staining and imaging conditions, defining central versus peripheral localization, collecting clinically annotated recurrent and non-recurrent cohorts, training classifiers or establishing thresholds, validating sensitivity/specificity/precision/recall, and confirming that the association holds in independent patient populations.
For claims 11-13, a PHOSITA would need to repeat this development and validation for breast cancer, prostate cancer, lung cancer, melanoma, kidney cancer, thyroid cancer, pancreatic cancer, stomach cancer, bladder cancer, DCIS, LCIS, ADH, ALH, and ipsilateral breast cancer recurrence. The specification does not provide working examples across these cancers and conditions.
For claims 15-17 and 19, a PHOSITA would also need to determine how the predicted recurrence result changes treatment. The PHOSITA would need to determine whether to use surgery, inhibitors to enzyme or transporter accumulation at plasma membrane, colchicine, taxol, calmodulin antagonists, prenylation inhibitors, anesthetics, immunotherapy, radiotherapy, or chemotherapeutic agents. The PHOSITA would need to determine appropriate doses, routes, schedules, combinations, patient-selection rules, treatment thresholds, safety profiles, and clinical endpoints. The specification provides no working treatment examples and no cancer-specific treatment protocols tied to the localization result beyond listing broad treatment categories and compounds.
Therefore, considering the Wands factors as a whole, the specification enables, at most, certain limited embodiments involving immunostaining/imaging of DCIS breast tissue samples and computer-assisted classification of PFKL, PFKFB4, and/or phospho-GLUT1 localization patterns in a limited DCIS recurrence dataset. The claims, however, encompass a much broader scope, including recurrence prediction and recurrent/non-recurrent cancer distinction across cancer generally, multiple cancer types, multiple biomarkers, peripheral intracellular localization criteria, and broad treatment regimens following prediction. Given the breadth of the claims, the limited working examples, the lack of generalizable guidance, the unpredictability reflected in the prior art, and the substantial experimentation required, claims 5-7, 11-13, 15-17, 19, and 20-21 are rejected under 35 U.S.C. 112(a).
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 8rejected 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 8 recites the limitation "immunostaining the sample with a primary antibody directed to the biomarker for cancer recurrence" in lines 3-4. There is insufficient antecedent basis for this limitation in the claim. Specifically, there is insufficient antecedent basis for “the biomarker for cancer recurrence.” Claim 1, from which claim 8 depends, recites determining intracellular localization of phosphofructokinase type L (PFKL) and/or phosphofructokinase/fructose-2,6-bisphosphatase type 4 (PFKFB4) in cells from the sample, but does not previously introduce “a biomarker for cancer recurrence.” Therefore, it is unclear whether “the biomarker for cancer recurrence” refers to PFKL, PFKB4, both PFKL and PFKFB4, or another biomarker. For purposes of compact prosecution, “the biomarker for cancer recurrence” will be interpreted as referring to phosphofructokinase type L (PFKL) and/or phosphofructokinase/fructose-2,6-bisphosphatase type 4 (PFKFB4), as recited in claim 1. Appropriate correction is required.
Claims 9 and 10 are rejected under 35 U.S.C. 112(b) for the same reasons because they depend from claim 8.
Claim 15 recites the limitation "treating a subject predicted to have cancer recurrence with surgery" in line 2. There is insufficient antecedent basis for this limitation in the claim. Specifically, there is insufficient antecedent basis for “a subject predicted to have cancer recurrence.” Claim 15 depends from claim 1, which recites obtaining a sample from a subject and determining intracellular localization of PFKL and/or PFKFB4 in cells from the sample, but claim 1 does not previously recite predicting that the subject has cancer recurrence or otherwise identify “a recurrence.” Therefore, it is unclear whether the “subject predicted to have cancer recurrence” is the same “subject” from whom the sample is obtained, and it is further unclear what claim step or limitation provides the prediction of cancer recurrence. For purposes of compact prosecution, “a subject predicted to have cancer recurrence” will be interpreted as referring to the subject from whom the sample is obtained in claim 1, wherein the prediction of cancer recurrence is based on the determined intracellular localization of PFKL and/or PFKFB4 in cells from the sample. Appropriate correction is required.
Claim 16 recites the limitation "treating a subject predicted to have cancer recurrence with administration of inhibitors to enzyme or transporter accumulation at plasma membrane" in lines 2-3. Specifically, there is insufficient antecedent basis for “a subject predicted to have cancer recurrence.” Claim 16 depends from claim 1, which recites obtaining a sample from a subject and determining intracellular localization of PFKL and/or PFKFB4 in cells from the sample, but claim 1 does not previously recite predicting that the subject has cancer recurrence or otherwise identify “a subject predicted to have cancer recurrence.” Therefore, it is unclear whether the “subject predicted to have cancer recurrence” is the same “subject” from whom the sample is obtained, and it is further unclear what claim step or limitation provides the prediction of cancer recurrence. For purposes of compact prosecution, “a subject predicted to have cancer recurrence” will be interpreted as referring to the subject from whom the sample is obtained in claim 1, wherein the prediction of cancer recurrence is based on the determined intracellular localization of PFKL and/or PFKFB4 in cells from the sample. Appropriate correction is required.
Claims 17 and 19 are rejected under 35 U.S.C. 112(b) for the same reasons because they depend from claim 16.
Claim 17 contains the trademark/trade name taxol. Where a trademark or trade name is used in a claim as a limitation to identify or describe a particular material or product, the claim does not comply with the requirements of 35 U.S.C. 112(b) or 35 U.S.C. 112 (pre-AIA ), second paragraph. See Ex parte Simpson, 218 USPQ 1020 (Bd. App. 1982). The claim scope is uncertain since the trademark or trade name cannot be used properly to identify any particular material or product. A trademark or trade name is used to identify a source of goods, and not the goods themselves. Thus, a trademark or trade name does not identify or describe the goods associated with the trademark or trade name. In the present case, the trademark/trade name is used to identify/describe an inhibitor to enzyme accumulation at the plasma membrane, and, accordingly, the identification/description is indefinite. Appropriate correction is required.
Claim 19 recites the limitation "wherein the treatment regimen comprises one or more of: surgery; administration of an inhibitor to enzyme or transporter accumulation at plasma membrane; immunotherapy; radiotherapy; and administration of a chemotherapeutic agent" in lines 1-4. Specifically, there is antecedent basis for “the treatment regimen.” Claim 19 depends from claim 16, which recites “treating a subject predicted to have cancer recurrence with administration of inhibitors to enzyme or transporter accumulation at plasma membrane,” but claim 16 does not previously introduce a “treatment regimen.” Therefore, it is unclear what prior claim limitation provides antecedent basis for “the treatment regimen.” Appropriate correction is required.
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 5-7, 11-13, and 20-21 are rejected under 35 U.S.C. 101 because the claimed invention is directed to a judicial exception, specifically a law of nature/natural phenomenon, i.e., the naturally occurring relationship between peripheral intracellular localization of one or more biomarkers, including phosphofructokinase type L (PFKL), phosphofructokinase/fructose-2,6-bisphosphatase type 4 (PFKFB4), and/or phosphorylated glucose transporter type 1 (pGLUT1), and cancer recurrence.
This rejection is made in accordance with Patent Subject Matter Eligibility as set forth in MPEP §2106. Analysis of subject-matter eligibility under 35 U.S.C. §101 requires consideration under these steps as followed:
Step 1 – Statutory Category (Refer to MPEP §2106.03): Claims 5-7, 11-13, and 20-21 are drawn to a process, which falls within a statutory category under 35 U.S.C. §101.
Step 2A, Prong One – Recitation of a Judicial Exception (Refer to MPEP §2106.04): Regarding claim 5, the claim recites that “peripheral intracellular localization of PFKL and/or PFKFB4 predicts cancer recurrence.” Thus, claim 5 recites a naturally occurring biological relationship/correlation between the intracellular localization pattern of PFKL and/or PFKFB4 in cells from a subject and the likelihood of cancer recurrence. This relationship between biomarker localization and cancer recurrence is a law of nature/natural phenomenon. Accordingly, claim 5 recites a judicial exception.
Regarding claim 6, the claim recites that “peripheral intracellular localization of PFKL, PFKFB4, and/or pGLUT1 predicts cancer recurrence.” Thus, claim 6 similarly recites a naturally occurring biological relationship/correlation between peripheral intracellular localization of PFKL, PFKFB4, and/or pGLUT1 and cancer recurrence. Accordingly, claim 6 recites a judicial exception.
Regarding claim 7, the claim recites that “peripheral intracellular localization of PFKL, PFKFB4, and/or the one or more additional biomarkers predicts cancer recurrence.” Thus, claim 7 recites a naturally occurring biological relationship/correlation between peripheral intracellular localization of biomarkers in cells from a subject and cancer recurrence. Accordingly, claim 7 recites a judicial exception.
Claims 11-13 depend directly or indirectly from claim 5 and therefore incorporate the judicial exception recited in claim 5. Claim 11 limits the cancer to selected cancer types, claim 12 further limits breast cancer to specific breast conditions, and claim 13 further limits the cancer recurrence to ipsilateral breast cancer recurrence. These dependent claims still rely on the naturally occurring relationship/correlation between peripheral intracellular localization of PFKL and/or PFKFB4 and cancer recurrence. Accordingly, claims 11-13 also recite a judicial exception.
Regarding claim 20, the claim recites “a method for distinguishing recurrent from non-recurrent cancer” comprising determining intracellular localization of a biomarker for cancer recurrence, wherein the biomarker comprises PFKL and/or PFKFB4, optionally comprises pGLUT1, and optionally comprises one or more additional biomarkers. Thus, claim 20 recites the naturally occurring biological relationship/correlation between intracellular localization of the recited biomarkers and whether cancer is recurrent or non-recurrent. Accordingly, claim 20 recites a judicial exception.
Regarding claim 21, the claim recites that “peripheral intracellular localization of the biomarker for cancer recurrence indicates recurrent cancer.” Thus, claim 21 expressly recites a naturally occurring biological relationship/correlation between peripheral intracellular localization of the biomarker and recurrent cancer. Accordingly, claim 21 recites a judicial exception.
Therefore, claims 5-7, 11-13, and 20-21 recite a judicial exception in the form of a law of nature/natural phenomenon.
Step 2A, Prong Two – Integration into a Practical Application (Refer to MPEP §2106.04 (d)): Regarding claim 5, the additional elements include obtaining a sample from a subject and determining intracellular localization of PFKL and/or PFKFB4 in cells from the sample. These additional elements do not integrate the judicial exception into a practical application. The steps of obtaining a biological sample and determining intracellular localization merely collect and observe biological information used to identify the naturally occurring relationship between peripheral intracellular localization of PFKL and/or PFKFB4 and cancer recurrence. The claim does not recite any particular unconventional staining, imaging, detection, or laboratory technique, nor does it recite an improvement to microscopy, immunostaining, biomarker detection technology, or another technical field. Rather, the claim uses routine sample analysis to observe the natural biomarker localization and then relies on the natural correlation to predict cancer recurrence. Thus, the additional elements amount to insignificant extra-solution activity and do not impose a meaningful limit on the judicial exception.
Regarding claims 6 and 7, the additional elements similarly include determining intracellular localization of PFKL, PFKFB4, pGLUT1, and/or one or more additional biomarkers. The inclusion of additional biomarkers does not integrate the judicial exception into a practical application because the claims still rely on the naturally occurring relationship between peripheral intracellular localization of the biomarkers and cancer recurrence. The claims do not improve a detection method, alter the sample, require a particular machine integral to the claim, or use the correlation to effect a particular treatment or prophylaxis. Rather, the claims merely expand the biomarkers observed as part of the same natural-correlation analysis.
Claims 11-13 depend directly or indirectly from claim 5 and further limit the cancer type or recurrence type. Claim 11 limits the cancer to selected cancers including breast cancer, prostate cancer, lung cancer, melanoma, kidney cancer, thyroid cancer, pancreatic cancer, stomach cancer, or bladder cancer. Claim 12 further limits the breast cancer to ductal carcinoma in situ of the breast, lobular carcinoma in situ, atypical ductal hyperplasia, or atypical lobular hyperplasia. Claim 13 further limits the cancer recurrence to ipsilateral breast cancer recurrence. These limitations do not integrate the judicial exception into a practical application because they merely limit the natural correlation to particular disease contexts or fields of use. The claims still use the observed peripheral intracellular localization of PFKL and/or PFKFB4 to predict cancer recurrence and do not recite an improvement to technology, a particular treatment step, a particular machine integral to the claim, or a meaningful transformation of an article.
Regarding claim 20, the additional elements include determining intracellular localization of a biomarker for cancer recurrence in a sample comprising cancer cells, wherein the biomarker comprises PFKL and/or PFKFB4, optionally comprises pGLUT1, and optionally comprises transketolase-like protein-1, glutathione synthetase, GTP-loaded RhoA, and/or RhoA. These elements do not integrate the judicial exception into a practical application. The claim is directed to distinguishing recurrent from non-recurrent cancer based on the intracellular localization of naturally occurring biomarkers in cancer cells. Determining intracellular localization is used merely to collect or observe information needed to apply the natural correlation. The claim does not recite an improved biomarker-detection technique, an improved imaging method, an unconventional sample-processing method, or any particular treatment or prophylaxis for recurrent cancer. The claim therefore merely applies the natural correlation in the field of cancer recurrence detection and does not integrate the exception into a practical application.
Regarding claim 21, the claim recites that peripheral intracellular localization of the biomarker for cancer recurrence indicates recurrent cancer. This limitation further states the natural correlation itself and does not add any additional element that integrates the judicial exception into a practical application. The claim does not require a treatment step, technological improvement, integral machine, or transformation beyond observing biomarker localization in cancer cells and interpreting that localization as indicating recurrent cancer.
Accordingly, claims 5-7, 11-13, and 20-21 do not integrate the judicial exception into a practical application. The additional elements amount to routine data-gathering, observation, and field-of-use limitations that merely identify and apply the naturally occurring relationship between biomarker localization and cancer recurrence. Therefore, the claims are directed to a judicial exception.
Step 2B, Inventive Concept (Refer to MPEP §2106.05): Here, the additional elements recited in claims 5-7, 11-13, and 20-21 include obtaining a biological sample, determining intracellular localization of PFKL, PFKFB4, pGLUT1 and/or other biomarkers, and limiting the correlation to particular cancer or recurrence contexts. These elements do not amount to significantly more than the judicial exception because the claims merely use conventional sample analysis, immunostaining/imaging-type detection, and biomarker localization observation to apply the natural relationship between biomarker localization and cancer recurrence.
The use of immunohistochemical staining of breast cancer tissue specimens to evaluate PFKFB4 expression was conventional in the field. Yao et al. (High Expression of Metabolic Enzyme PFKFB4 Is Associated with Poor Prognosis of Operable Breast Cancer. Cancer Cell International. Vol. 19. No. 1, June 2019) demonstrates that PFKFB4 was detected in breast cancer surgical specimens using immunohistochemistry, stating that “PFKFB4 expression was evaluated by immunohistochemistry in surgical specimens retrospectively collected from 200 patients with histologically proven invasive ductal breast cancer” (Abstract, p. 1). Yao further states that “archived and de-identified formalin-fixed paraffin-embedded (FFPE) samples were then analyzed” (Materials and methods, p. 2), and that “the TMAs were subjected to immunohistochemical staining for PFKFB4 using a two-step protocol (GTVision™ III)” (Materials and methods, p. 2). Also, Yao teaches determining intracellular localization of phosphofructokinase/fructose-2,6-bisphosphatase type 4 (PFKFB4) in cells from the sample, stating that “as shown in Fig. 1, positive staining for PFKFB4 protein was observed mainly in the cytoplasm of breast cancer cells, and most of the intra- or extra-tumor stromal cells were negative for PFKFB4” (Results, p. 3). Thus, evaluating intracellular localization of PFKFB4 in breast cancer tissue samples by routine immunohistochemistry was well-understood, routine, and conventional.
Likewise, the use of antibodies and imaging to determine intracellular or membrane localization of pGLUT1/GLUT1 was known in the art. Lee et al. (A Protein Kinase C Phosphorylation Motif in GLUT1 Affects Glucose Transport and Is Mutated in GLUT1 Deficiency Syndrome. Molecular Cell. Vol. 58, No. 5. June 2015 – IDS dated 01/09/2026) states that “for immunofluorescence, cells were plated on chamber slides (Lab-Tek), fixed in 4% paraformaldehyde, and permeabilized in 0.5% NP-40. Cells were stained with goat anti-GLUT1 (1:200; Santa Cruz) or anti-pGLUT1 S226 (1:200; Thermo) and secondary Alexa Fluor 488 or 546” (Experimental Procedures, p. 852). Lee further reports that “pGLUT1 (left column) shows a striking increase after TPA treatment with clear localization to cell membranes after TPA treatment” (Figure 3, p. 849). Therefore, antibody-based staining and imaging to observe intracellular or membrane localization of GLUT1/pGLUT1 was a conventional detection/localization activity.
Similarly, determining PFKL intracellular localization by fluorescence microscopy was known. Kohnhorst et al. (Identification of a Multienzyme Complex for Glucose Metabolism in Living Cells. Journal of Biological Chemistry. Vol. 292, No. 22, June 2017 - IDS dated 01/09/2026) states that “we have used wide-field and confocal fluorescence microscopy to investigate the spatial organization of metabolic enzymes participating in glucose metabolism in human cells” (Abstract, p. 9191). Kohnhorst further states that “human liver-type phosphofructokinase 1, tagged with a monomeric form of enhanced green fluorescent protein (PFKL-mEGFP), forms discrete cytoplasmic clusters of varying sizes in transfected HeLa cells (Fig. 1, A-C)” (Results, p. 9192). Kohnhorst also confirms use of antibody-based localization of endogenous PFKL, stating that “we performed immunocytochemistry against endogenous PFKL in HeLa and Hs578T cells” (Results, p. 9194). Thus, determining the intracellular localization of PFKL by fluorescence microscopy or immunocytochemistry was well-understood, routine, and conventional.
Taken together, Yao et al., Lee et al., and Kohnhorst et al. demonstrate that the additional activities recited or implicated by the claims—obtaining and analyzing samples, immunostaining biomarkers, using antibodies and detectable labels, imaging cells, and determining intracellular or membrane localization of PFKFB4, PFKL, and/or pGLUT1—were conventional laboratory and diagnostic activities known in the field. The claims do not recite an unconventional staining protocol, an improved imaging system, a new antibody format, a new sample-processing technique, or a technological improvement to biomarker localization analysis. Instead, the claims use routine localization/detection activities to observe naturally occurring biomarker localization and apply the natural correlation between that localization and cancer recurrence.
The additional limitations of claims 11-13 do not add an inventive concept because they merely limit the natural correlation to particular cancer types or recurrence contexts, including breast cancer and ipsilateral breast cancer recurrence. Such disease-context limitations are field-of-use limitations and do not transform the natural correlation into patent-eligible subject matter.
Likewise, claims 20 and 21 do not add significantly more because they recite distinguishing recurrent from non-recurrent cancer or indicating recurrent cancer based on intracellular/peripheral intracellular localization of the recited biomarkers. The claimed determination of biomarker localization is performed at a high level of generality using conventional observation/detection techniques, and the distinguishing or indicating step merely applies the natural correlation.
Accordingly, the additional elements in claims 5-7, 11-13, and 20-21, individually and as an ordered combination, amount to no more than well-understood, routine, and conventional laboratory activities previously known in the field. Therefore, the claims do not recite an inventive concept sufficient to amount to significantly more than the judicial exception.
Ultimately, claims 5-7, 11-13, and 20-21 are directed to a judicial exception, specifically a law of nature/natural phenomenon, and the additional elements do not integrate the exception into a practical application or amount to significantly more than the exception. Therefore, claims 5-7, 11-13, and 20-21 are patent ineligible under 35 U.S.C. § 101.
Claim Rejections - 35 USC § 102
In the event the determination of the status of the application as subject to AIA 35 U.S.C. 102 and 103 (or as subject to pre-AIA 35 U.S.C. 102 and 103) is incorrect, any correction of the statutory basis (i.e., changing from AIA to pre-AIA ) for the rejection will not be considered a new ground of rejection if the prior art relied upon, and the rationale supporting the rejection, would be the same under either status.
The following is a quotation of the appropriate paragraphs of 35 U.S.C. 102 that form the basis for the rejections under this section made in this Office action:
A person shall be entitled to a patent unless –
(a)(1) the claimed invention was patented, described in a printed publication, or in public use, on sale, or otherwise available to the public before the effective filing date of the claimed invention.
(a)(2) the claimed invention was described in a patent issued under section 151, or in an application for patent published or deemed published under section 122(b), in which the patent or application, as the case may be, names another inventor and was effectively filed before the effective filing date of the claimed invention.
Claims 1, 8, and 14 are rejected under 35 U.S.C. 102(a)(1) as being anticipated by Yao et al. (High Expression of Metabolic Enzyme PFKFB4 Is Associated with Poor Prognosis of Operable Breast Cancer. Cancer Cell International. Vol. 19. No. 1, June 2019).
Regarding claim 1, Yao teaches a method comprising obtaining a sample from a subject. Yao states, “clinical data and surgical specimens were retrospectively collected from 200 female patients who were diagnosed with stage I to III primary breast cancer at the Department of Breast Surgery in Fudan University Shanghai Cancer Center between January 2004 and January 2008. All specimens in this cohort were histologically confirmed with invasive ductal carcinoma and all participants underwent a mastectomy and axillary lymph node dissection or breast conservation surgery” (Materials and Methods, p. 2).
Yao teaches determining intracellular localization of phosphofructokinase/fructose-2,6-bisphosphatase type 4 (PFKFB4) in cells from the sample. Yao states, “PFKFB4 expression was evaluated by immunohistochemistry in surgical specimens retrospectively collected from 200 patients with histologically proven invasive ductal breast cancer” (Abstract, p. 1). Yao further states, “to investigate the clinical significance of PFKFB4 in breast cancer, we conducted immunohistochemical staining for PFKFB4 expression in the tumor samples” (Results, p. 3). Yao further states, “as shown in Fig. 1, positive staining for PFKFB4 protein was observed mainly in the cytoplasm of breast cancer cells, and most of the intra- or extra-tumor stromal cells were negative for PFKFB4” (Results, p. 3).
Regarding claim 8, Yao teaches that determining intracellular localization comprises immunostaining the sample with a primary antibody directed to the biomarker for cancer recurrence. Yao states, “as in our previous study, the tissue microarrays (TMAs) were subjected to immunohistochemical staining for PFKFB4 using a two-step protocol (GTVision™ III). PFKFB4 was detected using the rabbit anti-PFKFB4 polyclonal antibody [ab137785] (1:50;Abcam)” (Materials and Methods, p. 2). Also, Yao teaches imaging the sample. Yao states, “Fig. 1 Representative PFKFB4 immunohistochemical staining in breast cancer specimens in low-magnification (×100) and high-magnification (×400) images.” (Fig. 1, p. 4).
Regarding claim 14, Yao teaches that the sample comprises a formalin-fixed paraffin-embedded cancer tissue sample. Yao states, “tissue microarrays (TMAs) were constructed as previously described. Briefly, breast cancer specimens from the 200 surgical cases described above were fixed using standard protocols. Archived and de-identified formalin-fixed paraffin-embedded (FFPE) samples were then analyzed. After histological examination of the tissue samples by our dedicated pathologist, TMAs were developed by punching two 10-mm-diameter cores out of each tumor at two different sites” (Materials and Methods, p. 2).
Accordingly, Yao discloses, expressly or inherently, each and every limitation of claims 1, 8, and 14. Accordingly, claims 1, 8, and 14 are anticipated under 35 U.S.C. 102.
Claim Rejections - 35 USC § 103
In the event the determination of the status of the application as subject to AIA 35 U.S.C. 102 and 103 (or as subject to pre-AIA 35 U.S.C. 102 and 103) is incorrect, any correction of the statutory basis (i.e., changing from AIA to pre-AIA ) for the rejection will not be considered a new ground of rejection if the prior art relied upon, and the rationale supporting the rejection, would be the same under either status.
The following is a quotation of 35 U.S.C. 103 which forms the basis for all obviousness rejections set forth in this Office action:
A patent for a claimed invention may not be obtained, notwithstanding that the claimed invention is not identically disclosed as set forth in section 102, if the differences between the claimed invention and the prior art are such that the claimed invention as a whole would have been obvious before the effective filing date of the claimed invention to a person having ordinary skill in the art to which the claimed invention pertains. Patentability shall not be negated by the manner in which the invention was made.
The factual inquiries for establishing a background for determining obviousness under 35 U.S.C. 103 are summarized as follows:
1. Determining the scope and contents of the prior art.
2. Ascertaining the differences between the prior art and the claims at issue.
3. Resolving the level of ordinary skill in the pertinent art.
4. Considering objective evidence present in the application indicating obviousness or nonobviousness.
Claims 2-4 are rejected under 35 U.S.C. 103 as being unpatentable over Yao et al. in view of Lee et al. (A Protein Kinase C Phosphorylation Motif in GLUT1 Affects Glucose Transport and Is Mutated in GLUT1 Deficiency Syndrome. Molecular Cell. Vol. 58, No. 5. June 2015 – IDS dated 01/09/2026) and Li et al. (RhoA Protein Is Generally Distributed in the Nuclei of Cancer Cells. Oncology Reports. Vol. 24, No. 4, October 2010).
With respect to the teachings of Yao, see the discussion above, which applies equally here. However, Yao differs from the instant claim in failing to expressly teach or specify that the method further comprises determining intracellular localization of one or more additional biomarkers in the cells of the sample, wherein the one or more additional biomarkers includes phosphorylated glucose transporter type 1 (pGLUT1), transketolase-like protein-1, glutathione synthetase, GTP-loaded RhoA, and/or RhoA, as recited in claims 2-4.
However, Lee teaches determining intracellular localization of phosphorylated glucose transporter type 1 (pGLUT1). Lee states, “through in vitro kinase assays, mass spectrometry, and phosphospecific antibodies, we identify serine 226 in GLUT1 as a PKC phosphorylation site. Phosphorylation of S226 is required for the rapid increase in glucose uptake and enhanced cell surface localization of GLUT1 induced by the phorbol ester 12-O-tetradecanoyl-phorbol-13-acetate (TPA)” (Summary, p. 845). Lee further states, “phosphorylation of S226 increases GLUT1 membrane localization” (Highlights, p. 844). Lee further states, “immunofluorescence confirmed a clear localization of pGLUT1 S226 at the cell membrane in WT, but not S226A-expressing oocytes (Figure 2D)” (Results, p. 847). Lee further states, “pGLUT1 (left column) shows a striking increase after TPA treatment with clear localization to cell membranes after TPA treatment” (Fig. 3C, p. 849). Lee further states, “for immunofluorescence, cells were plated on chamber slides, fixed in 4% paraformaldehyde, and permeabilized in 0.5% NP-40. Cells were stained with goat anti-GLUT1 (1:200; Santa Cruz) or anti-pGLUT1 S226 (1:200; Thermo) and secondary Alexa Fluor 488 or 546” (Experimental Procedures, p. 852).
Li teaches determining intracellular localization of RhoA in cancer tissues and cancer cell lines. Li states, “the aim of this study was to elucidate the localization of RhoA in human cancer tissues and cancer cell lines. Immunohistochemistry and immunofluorescence microscopy were used to determine the localization of RhoA in cancer tissues and cell lines, respectively” (Abstract, p. 1005). Li further states, “immunofluorescence microscopy revealed that in all cell lines examined in this experiment (including SGC-7901, HeLa, A549 and SW480), RhoA was localized not only on the membrane, in the cytosol, but also in the nucleus” (Abstract, p. 1005). Li further states, “the distribution of target protein of the cells was analyzed by fluorescence microscopy” (Materials and Methods, p. 1005). Li further states, “DAB was to show the localization of RhoA protein” (Materials and Methods, p. 1006). Li further states, “immunohistochemistry staining (Fig. 5) of different cancer tissues including colon cancer, breast cancer, esophagus cancer and gastric cancer showed that the nuclear localization of RhoA was found in each type of cancer tissue” (Results, p. 1007). Li further states, “RhoA was localized in the membrane, cytosol and the nucleus of the tissues.” (Fig. 5, p. 1007).
Therefore, it would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to modify Yao’s method of determining intracellular localization of PFKFB4 in cells from a subject sample to add known additional localization readouts by further determining intracellular localization of additional biomarkers, including pGLUT1 as taught by Lee and RhoA as taught by Li, because Lee taught that phosphorylation of GLUT1 at S226 was a functionally significant modification that affected GLUT1 membrane localization and could be detected with phosphospecific antibodies and immunofluorescence, and Li taught that RhoA localization in cancer tissues and cancer cell lines could be determined by immunohistochemistry and immunofluorescence microscopy. A person of ordinary skill in the art would have been motivated to include these additional localization determinations because Yao already used immunohistochemical localization of a metabolic/prognostic biomarker in tissue samples, while Lee and Li taught that pGLUT1 and RhoA were additional intracellularly localized proteins whose membrane, cytosolic, and/or nuclear localization could be detected using routine antibody-based microscopy techniques. A person of ordinary skill in the art would have had a reasonable expectation of success in making this modification because the proposed modification merely applies known immunostaining/immunofluorescence localization techniques to additional known biomarkers using commercially available or expressly described antibodies, and Lee and Li demonstrated that such antibody-based methods successfully detected pGLUT1 and RhoA localization in cells/tissues.
Claims 9 and 10 are rejected under 35 U.S.C. 103 as being unpatentable over Yao et al. in view of Li et al.
With respect to the teachings of Yao, see the discussion above, which applies equally here. However, Yao differs from the instant claim in failing to expressly teach or specify that the primary antibody is detected with a secondary antibody comprising a detectable label, as recited in claim 9, or that imaging the sample comprises fluorescence microscopy, as recited in claim 10.
Li teaches detecting a primary antibody with a secondary antibody comprising a detectable label and imaging a sample by fluorescence microscopy to determine intracellular localization of a protein biomarker. As discussed above, Li states that “immunohistochemistry and immunofluorescence microscopy were used to determine the localization of RhoA in cancer tissues and cell lines, respectively” (Abstract, p. 1005). Li further states, “the cells grown on cover slips were fixed with freshly prepared paraformaldehyde (40 g/l in PBS) for 30 min. After being penetrated with 30 ml/l Triton X-100 and blocked with 30 g/l BSA, the cells were incubated with the primary antibody at 4˚C over night (o/n) and then with fluorescein isothiocyanate (FITC) or tetrarhodamine isothiocyanate (TRITC)-conjugated second antibody for 1 h at room temperature (RT), with three washes after each incubation. The distribution of target protein of the cells was analyzed by fluorescence microscopy” (Materials and Methods, p. 1005). Also, as discussed above, Li further states, “immunofluorescence microscopy revealed that in all cell lines examined in this experiment (including SGC-7901, HeLa, A549 and SW480), RhoA was localized not only on the membrane, in the cytosol, but also in the nucleus” (Abstract, p. 1005). Li further states, “Figure 1. Nuclear localization of RhoA in different cancer cell lines. Human gastric cancer cell line SGC-7901, human cervix cancer cell line HeLa, human colon adenocarcinoma cell line SW480 and human lung cancer cell line A549 were immunofluorescently stained with a specific mAb against RhoA and the results showed that in all cell lines, RhoA was localized on the membrane, in the cytosol, and mainly in the nucleus (original magnification, x400)” (Li, Fig. 1, p. 1006).
Therefore, it would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to modify Yao’s immunostaining/imaging method to detect the primary antibody with a secondary antibody comprising a detectable label and to image the sample by fluorescence microscopy as taught by Li, because Li expressly teaches an antibody-based localization method in which cells are incubated with a primary antibody and then with FITC- or TRITC-conjugated secondary antibody, followed by analysis of target-protein distribution using fluorescence microscopy. A person of ordinary skill in the art would have been motivated to make this modification because Yao and Li both concern antibody-based detection of intracellular localization/expression of cancer-associated protein biomarkers, and Li demonstrates that fluorescently labeled secondary-antibody detection is suitable for visualizing the membrane, cytosolic, and nuclear distribution of a protein target in cancer cells. A person of ordinary skill in the art would have recognized that using a FITC- or TRITC-conjugated secondary antibody in Yao’s antibody-based PFKFB4 staining method would provide a conventional detectable label for visualizing the bound primary antibody, and that fluorescence microscopy would be an established imaging modality for observing intracellular biomarker localization. A person of ordinary skill in the art would have had a reasonable expectation of success because labeled secondary antibodies and fluorescence microscopy were routine, predictable tools for immunostaining-based localization analysis, and Li shows successful use of those tools to determine intracellular localization of a cancer-associated protein biomarker. The proposed modification therefore would have involved the predictable use of a known labeled-secondary-antibody detection and fluorescence-imaging technique according to its established function in an otherwise analogous antibody-based biomarker-localization assay.
Ultimately, claims 2-4 and 9-10 are rejected under 35 U.S.C. 103 as being unpatentable over the applied prior art. The cited references collectively teach or suggest each of the additional limitations recited in these claims, and it would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to combine the teachings of the references with a reasonable expectation of success.
Conclusion
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/E.O./Examiner, Art Unit 1677
/BAO-THUY L NGUYEN/Supervisory Patent Examiner, Art Unit 1677 June 29, 2026
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