DETAILED ACTION
This Office action details a final action on the merits for the above referenced application No. Claims 7-13 are pending in this application.
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 Claims
Claims 1-6 are cancelled. Claims 7 and 10-11 are amended. Claim 13 is new.
Response to Amendment
The amendments filed on 28 Jul. 2026 have been entered.
Response to Arguments
In view of Applicants amendments, the objection to claim 10 because of minor informalities is withdrawn.
In view of Applicants amendments, the rejection of claims 7-12 under 35 USC 112(b) as being indefinite for failing to particularly point out and distinctly claim the subject matter is withdrawn.
In view of Applicants amendments, the rejection of claims 7-9 and 12 under 35 USC 102(a)(1) as being anticipated by Yin et al. (Sens. Act. B. Chemical; published 2019) is withdrawn.
In view of Applicants amendments, the rejection of claims 7-12 under 35 USC 103 as being unpatentable over Yin et al. (Sens. Act. B. Chemical; published 2019), in view of Valanciunaite et al. (Anal. Chem.; published 10 Mar. 2020) is withdrawn.
New Grounds of Rejection
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.
This application currently names joint inventors. In considering patentability of the claims the examiner presumes that the subject matter of the various claims was commonly owned as of the effective filing date of the claimed invention(s) absent any evidence to the contrary. Applicant is advised of the obligation under 37 CFR 1.56 to point out the inventor and effective filing dates of each claim that was not commonly owned as of the effective filing date of the later invention in order for the examiner to consider the applicability of 35 U.S.C. 102(b)(2)(C) for any potential 35 U.S.C. 102(a)(2) prior art against the later invention.
Claim(s) 7-13 is/are rejected under 35 U.S.C. 103 as being unpatentable over Yin et al. (Sens. Act.: B Chem.; published 2019), in view of Valanciunaite et al. (Anal Chem.; published 10 Mar. 2020) and Onodera et al. (US 2016/0011086 A1; published 14 Jan. 2016; see attached 892).
Yin et al. teach two-photon fluorescence imaging of lipid drops polarity toward cancer diagnosis in living cells and tissue (see title). Yin et al. teach that due to its excellent solvatochromism, the probe CBMC emits stronger fluorescence in normal cells but weaker in cancer cells. By means of CBMC, normal tissues with stronger fluorescence have been pointed out from cancerous tissues (see abstract). Increasing evidence suggests that the abnormality of LDs polarity is inseparable from neoplastic processes and lower polarity of LDs has been implicated in cancer cells. Accurately tracking LDs polarity is of great importance for the distinguishing between normal cells and cancer cells (pg. 251). The probe CBMC exhibits remarkable solvatochromic effects with polarity. In cancer cells, the probe CBMC displays weaker fluorescence owing to lower LDs polarity (pg. 252). Yin et al. teach fluorescence imaging in tumor tissues and normal organ tissues. With the help of a vibrating blade microtome, the slices were then cut to 200 mm thickness in 25 mM PBS (pH 7.4). After that, the slices were incubated with 15 µM CBMC in PBS buffer and bubbled with 95% O2 and 5% CO2 for 0.5 h at 37oC. After washed three times in PBS, they were transferred to the glass bottomed dishes and observed under TP confocal microscope (pg. 252). Yin et al. teach control and co-localization experiments of Nile Red and the probe CBMC for imaging 4T1 cells (see pg. 255). Yin et al. teach imaging of the tumor and normal organ slices pretreated with CBMC (15 µM) at the depth of 10-50 µm in TP mode (Fig. 6). The mean fluorescence intensity in the normal cells NHA is about 3 times higher than the cancer cells U87, which is suggested that the normal cells NHA could be obviously discriminated from cancer cells (pg. 257).
(Reads on a method for detecting tumor cells in a tissue comprising normal cells and tumor cells, the method using a three-dimensional tissue fragment derived from an organism; staining the cleared tissue with a fluorescent dye including a compound that exhibits solvatochromism; and detecting the tumor cells in the tissue and wherein the method is for use of inspection or diagnosis of a tumor and wherein the CBMC compound includes a fused polycyclic c conjugated structure having 3 ring and including at least one hydrophilic substituent containing at least one atom selected from nitrogen.)
With its outstanding solvatochromism, the variance of LDs polarity in different cancer cells and normal cells have been quantified making it suitable as an indicator for cancer diagnosis (pg. 257).
Yin et al. do not expressly teach the claimed method comprising a step for clearing the tissue. Yin et al. do not further teach 1-acetyl-6-piperidylpyrene represented by chemical formula (I), Nile Red, Laurdan, or di-4-aneppdhq. Yin et al. do not further teach a method wherein the absorption maximum wavelength of the compound is 300 to 600 nm in a 20 mM phosphate buffer at pH 7.4 and 25oC. Yin et al. do not further teach a method wherein the detecting of the tumor cells is performed by a multi-wavelength measurement in which fluorescence of two or more different wavelengths is detected and the respective intensities are integrated.
Valanciunaite et al. teach polarity mapping of cells and embryos by improved fluorescence solvatochromic pyrene probe (see title). Valanciunaite et al. teach that PA and PK show strong solvatochromism and an emission color response to lipid order in membranes. The PK probe enables high contrast polarity mapping of organelles including lipid droplets. Valanciunaite et al. teach the PK compound
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(see abstract). Valanciunaite et al. teach di-4-aneppdhq, Nile Red, and laurdan (pg. 6512). Valanciunaite et al. teach normalized absorption spectra of PA and PK in 20 mM phosphate buffer at pH 7.4 and an absorption maximum wavelength of 430 nm (Fig. 2). PK is highly sensitive to different lipid phases and to the presence of cholesterol. PK is clearly advantages as its absorption (400 nm) and emission (500-600 nm) are located in the visible spectral range (pg. 6514). PKL remains stable inside cells (pg. 6515). PK colocalized with Nile Red (Pg. 6515). PK shows sensitivity to environment polarity and lipid order (pg. 6516). Owing to the sensitivity of PK to environment polarity and lipid order, we applied it to map polarity distributions in cells by recording confocal imaging in two colors, green (531/40 nm) and Red (600/50 nm) and further merging them or making a red/green ratio-metric image (pg. 6516). PK showed advantageous spectroscopic properties as its absorption maximum matched well to common violet light sources around 400 nm and did not cross-talk with the blue laser commonly used for green markers (pg. 6518).
Onodera et al. teach a method for rendering tissue transparent, reagent for rendering tissue transparent and tissue observation method (see title). Onodera et al. teach that the present invention renders tissue transparent comprising a procedure of immersing the tissue in a water-soluble solvent having a refractive index of 1.4 to 1.7 ([0018], abstract). A technique is provided which can sufficiently render various organs transparent without causing their changes by a simpler operation without using any poisonous or dangerous organic chemicals ([0031]). Onodera et al. teach the observation of cancer. The transparentized cancer tissue is shown in FIG. 10. The 3 -dimensional fluorescence image of the cancer tissue is shown in A of Fig. 11. B of Fig. 11 shows a fluorescent observation image of a cancer tissue rendered transparent. For observation using the method for rendering tissue transparent according to the present invention (A), the observational depth limit was 1,000 µm or more and the distribution of tumor cells was identified (see example 7).
It would have been obvious to a person of ordinary skill in the art before the effective filing date to modify the method of Yin et al. (method for detecting tumor cells in a tissue comprising normal cells and tumor cells, the method comprising staining a three-dimensional tissue fragment from an organism with a fluorescent dye including a compound that exhibits solvatochromism and detecting the tumor and wherein the method is for inspection and diagnosis of a tumor) so that the method includes a step for clearing the three-dimensional tissue fragment derived from an organism as taught by Valanciunaite et al. and Onodera et al. because the clearing would have been expected to advantageously enable making the tissue transparent enabling enhanced imaging especially at greater depths. It would have been obvious to a person of ordinary skill in the art before the effective filing date to further modify Yin et al. by further substituting the CBMC fluorescent dye with 1-acetyl-6-piperidylpyrene of instant formula I, Nile Red, Laurdan, or di-4-aneppdhq as taught by Valanciunaite et al. because those solvochromic dyes would have been expected to provide equivalent solvochromic dyes enabling detecting cancer associated polarity changes in LDs and because the 1-acetyl-6-piperidinylpyrene that exhibits a maximum wavelength of about 400 nm in 20 mM phosphate buffer at pH 7.4 and room temperature (25oC) would have been expected show advantage spectroscopic properties. It would have been obvious to a person of ordinary skill in the art before the effective filing date to further modify the method of Yin et a. so the detecting of the tumor cell is performed by a multi-wavelength measurement in which fluorescence of two or more different wavelengths is detected and the respective fluorescence intensities are integrated as taught by Valanciunaite et al. because it would have been expected to enable mapping polarity distributions in a cell and because it would have been expected to advantageously take into account the sensitivity of the dye to environmental polarity.
Applicants Arguments
Applicants assert that Yin fails to disclose or suggest a method for detecting tumor cells in a three-dimensional tissue including normal cells and tumor cells wherein the method includes a tissue clearing step. Valanciunaite does not cure the deficiencies of Yin at least it does not disclose or suggest a method for detecting tumor cells in a three-dimensional tissue comprising normal cells and tumor cells wherein the method includes a tissue clearing step. As demonstrated in examples 2-4, the staining can distinguish and detect tumor cells from various normal cells. Using two photon microscopes, three-dimensional PK stained images were successfully obtained without thinly slicing even for tissues as thick as 500 µm.
Applicant's arguments filed 28 Jul. 2026 have been fully considered but they are not persuasive. Yin discloses and suggest a method for detecting tumor cells in a three-dimensional tissue fragments (200 mm thickness) comprising normal cells and tumor cells wherein the method comprises staining the tissue with the fluorescent dye including a compound that exhibits solvatochromism (CBMC) and detecting the tumor cells in the tissue. Fig. 6 provides images of the tumor and normal organ slices treated with CBMC at depths of 10-50 µm. Yin differs from claim 7 as amended because Lin does not teach clearing the three-dimensional tissue fragments prior to the staining. However, Onodera teaches and makes obvious tissue staining methods that include a step for clearing the tissue wherein the tissue is a three-dimensional tissue fragment derived from an organism. Onodera teaches that tissue clearing step has certain advantages including sufficiently rendering various organs transparent without causing their changes. Onodera provides for a method of observing cancer wherein the tissue clearing step enabled an observation depth of 1,000 µm or more. A recognized advantage is the strongest reason to combine. It would have been obvious to a person of ordinary skill in the art before the effective filing date to modify the tissue staining method of Yin so that the tissue staining method includes a step for clearing the tissue as taught by Onodera because the tissue clearing step would have been expected to advantageously enable rendering the tissue more transparent, reducing background noise and allow for greater observational depths.
Conclusion
Applicant's amendment necessitated the new ground(s) of rejection presented in this Office action. Accordingly, THIS ACTION IS MADE FINAL. See MPEP § 706.07(a). Applicant is reminded of the extension of time policy as set forth in 37 CFR 1.136(a).
A shortened statutory period for reply to this final action is set to expire THREE MONTHS from the mailing date of this action. In the event a first reply is filed within TWO MONTHS of the mailing date of this final action and the advisory action is not mailed until after the end of the THREE-MONTH shortened statutory period, then the shortened statutory period will expire on the date the advisory action is mailed, and any nonprovisional extension fee (37 CFR 1.17(a)) pursuant to 37 CFR 1.136(a) will be calculated from the mailing date of the advisory action. In no event, however, will the statutory period for reply expire later than SIX MONTHS from the mailing date of this final action.
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/SEAN R. DONOHUE/
Examiner, Art Unit 1618
/Robert A Wax/Supervisory Patent Examiner, Art Unit 1615
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