P53 amyloid-binding composition, method, and kit thereof

Abstract

p53 amyloid-binding composition, method, and kit thereof are disclosed in various embodiments herein. In one embodiment, the p53 amyloid-binding composition is used for the detection and localization of p53 amyloid aggregates and co-aggregates in cancer cells or tissues. The embodiments herein also disclose a method for identifying and differentiating histopathological grades of cancer using the p53 amyloid-binding composition. The method, according to embodiments herein, is a low-cost, efficient, sensitive, and versatile method of staining for the accurate indication of p53 amyloids in cancer cells or tissues, facilitating early detection and accurate assessment of cancer progression. Embodiments herein further disclose a kit for the identification and differentiation of oral and stomach cancers and grades thereof.

Description

“p53 amyloid-binding composition, method, and kit thereof’CROSS REFERENCE TO RELATED APPLICATIONThis application is based on and derives the benefit of Indian Complete Application 202421031922, the contents of which are incorporated herein by reference.TECHNICAL FIELD

[0001] Embodiments disclosed herein relate to amyloid-binding composition and more particularly p53 amyloid-binding composition for the identification of cancer proteins. Further, the embodiments particularly relate to a kit and a method of identification and differentiation of cancer using p53 amyloid- binding composition.BACKGROUND

[0002] Cancer is a global health challenge affecting millions of people. It stands as the second most common cause of death globally, contributing to one out of every six fatalities. Despite significant advancements in medical research and treatment options, cancer remains a formidable health concern worldwide.

[0003] Detecting cancer in its early stages increases the likelihood of survival significantly, as early intervention allows for surgical removal or the application of less intensive drug treatments. The average 5-year survival rate at the early stage is 91%, in contrast to the average 5-year survival rate at the late stage, which is 26%.

[0004] p53 is one of the most frequently mutated genes in human cancers. As a result, p53 has garnered significant attention as a potential cancer biomarker. p53 (also known as TP53) is a DNA sequence- specific transcription factor that suppresses tumor growth by regulating a large number of genes involved in cell cycle arrest, DNA repair, senescence, apoptosis, cell motility, adhesion, and migration. The presence of mutations or abnormalities in the p53 gene, as well as alterations in p53 protein expression levels, can serve as indicators of oncogenic processes within the body.

[0005] Mutant p53 displays its loss of tumor suppressor functions and demonstrates a gain of oncogenic properties. The mutant p53 displays gain-of-function by interacting with wild-type (WT) p53, p53 -related proteins p63 and p73 (also known as TP63 and TP73, respectively), and other transcription factors. p53 aggregation and amyloid formation are also correlated with the loss of p53 tumorsuppressive function and the gain of oncogenic function. Further, p53 amyloids have been observed to show prion-like properties in cells and can induce cancerous transformation in normal cells.

[0006] Although p53 aggregation has been shown in various cancers, the utilization of the aggregated / amyloid state of p53 for cancer tissue detection is not known.

[0007] The current state-of-the-art method for detecting p53 aggregates / amyloid includes using techniques such as Fluorescence Microscopy, Immunohistochemistry (IHC), Electron Microscopy (EM), Thioflavin-T Assay, Filter Trap Assay, Atomic Force Microscopy (AFM), Protein Aggregation Assays, and Mass Spectrometry. However, these methods are hampered by various drawbacks including limited sensitivity, complex sample preparation, cross reactivity, quantification challenges, invasive sample processing, limited tissue penetration, inability to distinguish between amyloid subtypes, high cost, time consuming protocols and so on.

[0008] Hence, there is a need in the art for suitable p53 amyloid -binding detection methods or compositions that may facilitate the detection of amyloid- like p53 for the detection of cancers as well as overcome the above-mentioned drawback(s).OBJECTS

[0009] The principal object of embodiments herein is to disclose p53 amyloid-binding composition that enables the detection and localization of p53 amyloid aggregates and co-aggregates in cancer cells or tissues.

[0010] Another object of the embodiments herein is to disclose p53 amyloid-binding composition including Thio S along with p53 antibody for p53 amyloid detection in cancer tissues.

[0011] Another object of the embodiments herein is to disclose p53 amyloid-binding composition including amyloid- specific antibodies along with p53 antibodies for cancer tissue detection.

[0012] Another object of the embodiments herein is to disclose p53 amyloid-binding composition that has high sensitivity in detecting p53 amyloid aggregates and co-aggregates.

[0013] Another object of the embodiments herein is to disclose p53 amyloid-binding composition for immunofluorescence imaging to quantify p53 amyloid aggregates and co-aggregates.

[0014] Another object of the embodiments herein is to disclose p53 amyloid-binding composition that enable the identification and differentiation of histopathological grades of cancer.

[0015] Another object of the embodiments herein is to disclose a method for identifying at least one protein from the cancer proteins using a specific amyloid-binding composition.

[0016] Another object of embodiments herein is to disclose a method for the detection and identification of Oral (Grade I, II, III, and IV) and Stomach (Grade II and III) cancers based on p53 amyloid aggregates or co-aggregates.

[0017] Another object of the embodiments herein is to disclose a low-cost, efficient, sensitive, and versatile method of staining for accurate indication of p53 amyloids in cancer cells or tissues, facilitating early detection and accurate assessment of cancer progression.

[0018] Another object of the embodiments herein is to disclose a kit (Thio S solution) for the identification and differentiation of oral and stomach cancers and grades thereof.

[0019] These and other aspects of the embodiments herein will be better appreciated and understood when considered in conjunction with the following description and the accompanying drawings. It should be understood, however, that the following descriptions, while indicating at least one embodiment and numerous specific details thereof, are given by way of illustration and not of limitation. Many changes and modifications may be made within the scope of the embodiments herein without departing from the spirit thereof, and the embodiments herein include all such modifications.BRIEF DESCRIPTION OF FIGURES

[0020] Embodiments herein are illustrated in the accompanying drawings, throughout which like reference letters indicate corresponding parts in the various figures. The embodiments herein will be better understood from the following description with reference to the following illustratory drawings. Embodiments herein are illustrated by way of examples in the accompanying drawings, and in which:

[0021] FIGs. 1 A, IB and 1C are double immunofluorescence images using anti-p53 (DO-1) and anti-amyloid (OC) antibody depicting p53 colocalization with amyloids; wherein FIG. 1A depicts images of oral grade I; FIG. IB depicts images of oral grade II; FIG. 1C depicts images of oral grade III and IV; the yellow areas denote the colocalization of p53 and OC signals, the colocalized areas are shown with a white arrowhead, and different oral cancer grades are highlighted in different color boxes or lines: oral grade III (blue) and oral grade IV (red). Scale bars: 50 pm. Image representative of n=2 experiments, according to embodiments disclosed herein;

[0022] FIG. 2 are double immunofluorescence images using anti-p53 (DO- 1) and anti-amyloid (OC) antibodies depicting p53 colocalization with amyloids. The yellow areas denote the colocalization of p53 and OC signals and are shown with a white arrowhead. Representative images of stomach grade II and stomach grade III are shown. The different stomach cancer grades are highlighted in different color boxes: stomach grades II (red) and III (blue). Scale bar: 50 pm. Image representative of n=2 experiments, according to embodiments as disclosed herein;

[0023] FIGs. 3A and 3B are graphical representations depicting the areas with colocalized p53- and amyloid- specific OC antibody staining quantified using ImageJ, wherein 3A depicts the percentage of colocalized area for oral cancer and FIG. 3B depicts the percentage of colocalized area for stomach cancer. The values are plotted as mean+s.e.m., n=3 independent experiments, according to embodiments as disclosed herein;

[0024] FIG. 4 are immunofluorescence images depicting p53 amyloids in oral (O1-O4) and stomach cancer tissues (S2, S3) using an anti-p53 antibody (DO- 1) and amyloid-specific dye, ThioS. Scale bars are 50 pm. Images arerepresentative of 3 independent experiments, according to embodiments as disclosed herein;

[0025] FIG. 5 are double immunofluorescence images depicting an anti- p53 antibody (DO-1) and oligomer- specific antibody (Al l) in oral (01, 03) and stomach cancer tissues (S2, S3). Scale bars are 50 pm. Images are representative of 2 independent experiments, according to embodiments as disclosed herein;

[0026] FIG. 6A are double immunohistochemistry staining images (using anti-p53 antibody, DO-1, and anti-p73 antibody) depicting colocalization of p53 and p73 signals in oral cancers of different grades (01, 03) and stomach cancer (S2, S3). Images shown are from n=3 independent experiments, according to embodiments as disclosed herein;

[0027] FIG. 6B are double immunohistochemistry staining images (using anti-p53 antibody, DO-1, and anti-p63 antibody) depicting colocalization of p53 and p63 signals in oral cancers (01, 03) and stomach cancer (S2, S3). Image representative of n=3 experiments, according to embodiments as disclosed herein;

[0028] FIGs. 7A and 7B are graphical representations depicting the quantification of p53 / p73 colocalization using image J, wherein FIG. 7A depict oral tissue grades; and FIG. 7B depicts stomach tissue grades, respectively. The values are plotted as mean ± s.e.m., n=2 independent experiments, according to embodiments as disclosed herein;

[0029] FIGs. 8A and 8B are graphical representations depicting the quantification of p53 / p63 colocalization using image J, wherein FIG. 8A depict oral tissue grades; and FIG. 8B depicts stomach tissue grades, respectively. The values are plotted as mean ± s.e.m., n=2 independent experiments, according to embodiments as disclosed herein;

[0030] FIG. 9A is a correlation plot depicting a positive correlation between the percentage of p53 / p73 colocalization with oral cancer grades, according to embodiments as disclosed herein;

[0031] FIG. 9B is a plot depicting a positive correlation between the percentage of p53 / p63 colocalization with oral cancer grades, according to embodiments as disclosed herein;

[0032] FIGs. 10A and 10B are western blot images depicting p53 expression in the soluble and insoluble fraction, wherein FIG. 10A depicts oral tissues, and FIG.10B depicts stomach tissues, respectively. (GAPDH - loading control). Image representative of n=2 experiments, according to embodiments as disclosed herein;

[0033] FIGs. 10C and 10D are graphical representations depicting the quantification of p53 band intensity using Image J, wherein FIG. 10C depicts oral tissues and FIG. 10D depicts stomach tissues, respectively. (Insol T-insoluble fraction of the tumor tissue lysate; Sol T- soluble fraction of the tumor tissue lysate; Insol N - insoluble fraction of the normal tissue lysate and Sol N- soluble fraction of the normal tissue lysate), according to embodiments as disclosed herein;

[0034] FIG. 11A is an image depicting western blot analysis of immunoprecipitated p53 from the stomach and oral cancer tissues using p73 and p63 antibodies, according to embodiments as disclosed herein;

[0035] FIG. 11B is an image depicting western blot analysis of immunoprecipitated p73 and p63 from the stomach and oral cancer tissues using p53 antibody, according to embodiments as disclosed herein;

[0036] FIG. 12A is a dot blot depicting amyloid content and p53 expression in different grades of oral (01 to 04) and stomach cancer tissues (S2 and S3). Image representative of n=2 experiments, according to embodiments as disclosed herein;

[0037] FIGs. 12B and 12C are graphical representations depicting the quantification of p53 amyloid load using OC antibody using Image J in different cancer grades, wherein FIG. 12B depicts oral tissues, and 12C depicts stomach tissues, respectively. The values were plotted as mean ± s.e.m., n=2 independent experiments, according to embodiments as disclosed herein;

[0038] FIG. 13A is an immunoelectron microscopy image depicting 10 nm gold particle decorations on the fibrils due to the presence of p53 in fibrils isolated from oral and stomach cancer tissues. Scale bars, 200 nm. Images representative of n=3 independent experiments according to embodiments as disclosed herein;

[0039] FIG. 13B is a double immunoelectron microscopy image showing 5 nm (gold-labeled secondary antibody against primary antibody of p53) and 10nm gold (gold-labeled secondary antibody against primary antibody of p63) particle decorations on the fibrils due to the presence of p53 and p63 coaggregation in fibrils isolated from grade III oral cancer tissues. Scale bars, 200 nm. Image representative of n=2 experiments, according to embodiments as disclosed herein;

[0040] FIGs. 14A and 14B are FTIR spectra depicting amyloid fibrils isolated from cancer biopsies, wherein FIG. 14A depicts FTIR spectra of oral tissue, and FIG. 14B depicts FTIR spectra of stomach cancer tissues, respectively, according to embodiments as disclosed herein;

[0041] FIGs. 15A and 15B are 3D surface plots depicting the amyloid load (b-sheet rich amyloid region (1640-1620 cm1) analyzed from FTIR imaging, wherein FIG. 15A depicts different grades of oral tissue, and FIG. 15B depicts different grades of stomach tissues. The corresponding FTIR signature is shown for each of the oral / stomach tumor tissue grades. The values were plotted as mean ± s.e.m., n=2 independent experiments, according to embodiments disclosed herein.DETAILED DESCRIPTION

[0042] The embodiments herein and the various features and advantageous details thereof are explained more fully with reference to the non-limiting embodiments that are illustrated in the accompanying drawings and detailed in the following description. Descriptions of well-known components and processing techniques are omitted so as to not unnecessarily obscure the embodiments herein. The examples used herein are intended merely to facilitate an understanding of how the embodiments herein may be practiced and to further enable those of skill in the art to practice the embodiments herein. Accordingly, the examples should not be construed as limiting the scope of the embodiments herein.

[0043] For the purposes of interpreting this specification, the definitions (as defined herein) will apply, and whenever appropriate, the terms used in singular will also include the plural and vice versa. It is to be understood that the terminology used herein is for the purposes of describing particular embodiments only and is not intended to be limiting. The terms “comprising”, “having” and “including” are to be construed as open-ended terms unless otherwise noted.

[0044] The words / phrases "exemplary", “example”, “illustration”, “in an instance”, “and the like”, “and so on”, “etc.”, “etcetera”, “e.g.,”, “i.e.,” are merely used herein to mean "serving as an example, instance, or illustration." Any embodiment or implementation of the present subject matter described herein using the words / phrases "exemplary", “example”, “illustration”, “in an instance”, “and the like”, “and so on”, “etc.”, “etcetera”, “e.g.,”, “i.e.,” are not necessarily to be construed as preferred or advantageous over other embodiments.

[0045] The embodiments herein disclose p53 amyloid-binding composition for identification of cancer protein aggregates or co-aggregates, comprising: at least one p53 specific antibody; and at least one agent selected from a group consisting of at least one amyloidspecific dye and at least one amyloid-specific antibody; wherein the p53 amyloid-binding composition binds with at least one cancer protein aggregate or co-aggregate associated with oral and stomach cancers and grades thereof.

[0046] A method for identification of cancer protein aggregates or coaggregates using the p53 amyloid-binding composition is also provided in various embodiments herein. Embodiments herein further disclose a kit for the identification and differentiation of oral and stomach cancers and grades thereof.

[0047] The invention utilizes the unique ability of the p53 amyloid -binding composition to selectively bind to cancer proteins, especially amyloid aggregates and co-aggregates of p53, p63, p73, and isoforms thereof, that are present in cancerous tissues. By quantifying the p53 amyloid load using immunofluorescence, the invention enables the detection and localization of cancer cells and cancer grade, facilitating early diagnosis and accurate assessment of cancer progression. Amyloid aggregates are typically associated with neurodegenerative diseases. However, p53 amyloids have been increasingly recognized as potential cancer biomarkers. The present invention offers p53 amyloid staining with low-cost dyes and amyloid- specific antibodies that can detect the cancer tissue irrespective of wild-type or mutant status.

[0048] The term “p53 amyloid-binding composition”, as used herein, refers to a combination of antibodies and dye that specifically identifies only p53amyloid forms. In one embodiment, the p53 amyloid-binding composition includes a combination of at least one p53 specific antibody and at least one amyloid-specific dye. In one other embodiment, the p53 amyloid-binding composition includes a combination of at least one p53 specific antibody and at least one amyloid- specific antibody.

[0049] In one embodiment, the p53 amyloid-binding composition specifically binds to at least one protein from the p53 protein family. In one embodiment, the p53 amyloid-binding composition specifically bind to p53 amyloid aggregates and its' co-aggregates with p63. In one other embodiment, the p53 amyloid-binding composition specifically bind to p53 amyloid aggregates and its' co-aggregates with p63 or p73. In one embodiment, the p53 amyloid -binding composition facilitate the detection of p53 amyloid aggregate and its co-aggregates with p63 or p73 and their potential correlation with cancer severity and progression. In one embodiment, the p53 amyloid-binding composition targets the protein amyloid backbone of p53 family proteins, enabling the detection of cancer- related protein aggregations.

[0050] In one embodiment, the p53 amyloid-binding composition includes at least one p53 specific antibody. Non-limiting examples of p53 specific antibodies include mouse monoclonal anti-human p53 protein DO-1, DO-7 (sc- 47698), P53 (DO-7) (MA5-12557), p53 (FL-393) (sc-6243), Pab 1801 (SC-98), PAb 240 (OP43), BP53-12 (ADI-KAP-PK035-D), and so on. In one embodiment, the at least one p53 specific antibody is anti-human p53 protein DO-1.

[0051] In one embodiment, the p53 amyloid-binding composition includes at least one amyloid-specific dye. Non-limiting examples of amyloid-specific dyes include fluorescein, cyanine, thioflavin T (Thio T), thioflavin S (Thio S), Congo red, m-I-Stilbene, Chrysamine G, 2-(4'-Methylaminophenyl) benzothiazole (BTA- 1), PIB, BF-227, X-34, TZDM, FDDNP, MeO-X-04, NIAD 4, Amylo-Glo, Nile red, and combinations thereof. In one embodiment, the at least one amyloidspecific dye is Thio-S.

[0052] In one embodiment, the p53 amyloid-binding composition includes at least one amyloid-specific antibody. Non-limiting examples of amyloid-specific antibodies include rabbit polyclonal oligomer- specific Al l, amyloid- specific (OC) antibody, and combinations thereof. In one embodiment, the at least oneamyloid-specific antibody is amyloid- specific antibody OC. In one other embodiment, the at least one amyloid-specific antibody is rabbit polyclonal oligomer- specific Al l.

[0053] The term “cancer proteins”, as used herein, refers to the “altered form of p53 protein family”, including three proteins: p53, p63, p73, and isoforms thereof. In one embodiment, the cancer proteins further include various structural forms including, but not limited to, monomeric (misfolded), oligomeric, amyloid structure, fibrillar form, aggregate forms, co-aggregate forms, folded, unfolded, misfolded forms of p53, p63, p73, and isoforms thereof. In one embodiment, the cancer proteins include p53 aggregates and co-aggregates. In one embodiment, the amyloid-binding composition is capable of binding with exposed amino acid residues of the cancer proteins, i.e., p53, p63, p73, or isoforms thereof.

[0054] p53 or TP53 is a DNA sequence- specific transcription factor that suppresses tumor growth by regulating a large number of genes involved in cell cycle arrest, DNA repair, senescence, apoptosis, cell motility, adhesion, and migration. p53 aggregation and amyloid formation are correlated with the loss of p53 tumor- suppressive function and the gain of oncogenic function. Amyloids are abnormal aggregates of proteins characterized by a fibrillar morphology of typically 7-13 nm in diameter and a P-sheet secondary structure. They are insoluble and tend to form fibrils, which are elongated thread-like structures. Amyloid formation is associated with several diseases, including Alzheimer's disease, Parkinson's disease, and type 2 diabetes. Although studies suggest that p53 amyloids might be associated with cancer, the relationship between the extent of p53 amyloid formation and cancer disease severity (such as cancer grade) is still not established.

[0055] The inventors of the disclosed innovation have successfully determined the correlation between the degree of p53 amyloid formation and the severity of cancer diseases, such as cancer grades including oral (Grade I, II, III, and IV) and stomach (Grade II and III). These amyloid aggregates are associated with loss of normal protein function and toxic gain-of-function.

[0056] p53, according to embodiments herein, includes wild type p53, and various isoforms including, but not limited to, p53a, p53p, p53y, A40p53a,A40p53p, A40p53y, A133p53a, A133p53p, A133p53y, A160p53a, A160p53p, and A160p53y.

[0057] p63 (also known as TP63) is a transcription factor of the p53 family. Unlike p53, p63 exhibits diverse functions, including the regulation of epithelial development, stem cell maintenance, and cell fate determination. Through its isoforms, including TAp63 and ANp63, p63 governs intricate gene expression programs involved in epithelial morphogenesis, stratification, and barrier formation. Moreover, p63 plays a pivotal role in maintaining genomic stability, orchestrating DNA damage responses, and modulating cellular senescence. Its aberrant expression and genetic alterations contribute to tumorigenesis, tumor progression, and therapeutic resistance, highlighting p63's significance as a potential diagnostic and prognostic marker in cancer.

[0058] p63, according to embodiments herein, includes wild type p63, and various isoforms including, but not limited to, TAp63a, TAp63b, TAp63c, DNp63a, DNp63b and DNp63cTAp63 and ANp63.

[0059] p73 is a tumor suppressor belonging to the p53 family of transcription factors. Through its transcriptional activity, p73 governs a wide array of cellular processes, including cell cycle regulation, DNA repair, and apoptosis induction. Notably, p73's ability to induce apoptosis in response to cellular stress mirrors p53's tumor-suppressive function, making it a key player in maintaining genomic stability and preventing tumorigenesis. However, unlike p53, which is frequently mutated in cancers, p73 mutations are relatively rare.

[0060] p73, according to embodiments herein, includes wild type p73, and various isoforms including, but not limited to, TAp73a, TAp73p, TAp73y, TAp735, TAp73s, TAp73(^, TAp73q, ANp73a, ANp73p and ANp73y isoforms.

[0061] When p53 forms amyloids, which are aggregates of misfolded proteins, it appears to have an affinity for binding to p63 and p73, effectively sequestering them. This sequestration can potentially disrupt the normal functions of p63 and p73, which are involved in regulating processes such as cell growth, differentiation, and apoptosis (programmed cell death). Therefore, the sequestration of p63 and p73 by p53 amyloids may have significant implications for cellular function and could contribute to pathological conditions associated with dysregulated p53 activity.

[0062] Embodiments herein further include a method for identification of cancer protein aggregates or co-aggregates using the p53 amyloid-binding composition as disclosed in various embodiments herein.

[0063] In one embodiment, the method includes: obtaining at least one tissue sample from an individual; contacting the sample with the p53 amyloid- binding composition; and detecting the degree of colocalization of the p53 amyloid-binding composition with at least one cancer protein; wherein the cancer protein is selected from a group consisting of p53, p63, p73 and isoforms thereof.

[0064] The term “tissue sample”, as used herein, includes samples having or suspected of having cancer. In one embodiment, the sample includes samples from oral cancer. In one other embodiment, the term sample includes samples from stomach cancer. In one embodiment, the term sample includes, samples having the cancer proteins, i.e., p53 protein family, wherein the p53 protein family members, i.e., p53, p63, and p73, may be present in their monomeric, oligomeric, amyloid structure, fibrillar form, aggregate forms, co-aggregate forms, folded, unfolded, and misfolded forms of p53, p63, p73, and isoforms thereof.

[0065] The sample includes oral, or stomach tissue obtained from an individual by surgical procedure through methods generally known in the field. Techniques for the acquisition of such samples are well-known in the art. The tissue sample, after collection, may be used in processed or unprocessed form. Processing of tissue samples may be performed according to methods known in the field. Processed tissue samples include, but are not limited to, frozen, flash- frozen, fresh, and paraffin fixed-embedded tissue samples.

[0066] The term “individual”, as used herein, includes any individual having or suspected of having cancer. In one embodiment, the individual may be having or suspected of having oral cancer. In one other embodiment, the individual may be having or suspected of having stomach cancer. In one embodiment, the individual is a mammal, such as a human.

[0067] In one embodiment, the method is also used to identify and differentiate histopathological grades of oral and stomach cancer.

[0068] In one embodiment, the p53 amyloid-binding composition can be used to identify and differentiate oral cancer of different histopathological grades. The disclosed amyloid-binding composition may be used to identify ordifferentiate grades of oral cancer as per WHO’s grading based on histopathological features, namely: Grade I (Well-differentiated), Grade II (Moderately differentiated), Grade III (poorly differentiated) and Grade IV (Undifferentiated). Accordingly, a method for identification and differentiation of Grade I, Grade II, Grade III, and Grade IV oral cancer is disclosed in various embodiments herein. Furthermore, the method can also be used to determine the prognosis of oral cancer. In one embodiment, the method is used for identifying and differentiating Grade I, Grade II, Grade III and Grade IV oral cancer.

[0069] In one other embodiment the p53 amyloid-binding composition can be used for detection and identification of stomach cancer of different histopathological grades. The disclosed amyloid-binding composition may be used to identify and differentiate grades of stomach cancer as per WHO’s grading based on histopathological features, namely: Grade I (Well-differentiated), Grade II (Moderately differentiated), Grade III (poorly differentiated) and Grade IV (Undifferentiated). Accordingly, a method for the identification and differentiation of Grade-I, Grade-II, and Grade-Ill stomach cancer is disclosed in various embodiments herein. Furthermore, the method can also be used in the prognosis of stomach cancer. In one embodiment, the method is used to identify and differentiate between Grade II and Grade III stomach cancer.

[0070] The p53 amyloid-binding composition, as disclosed in various embodiments herein, can be used in detection methods including, but not limited to, Thioflavin fluorescence assay, western blot, dot blot, immunoassays, immunostaining, enzyme-linked immunosorbent assay (ELISA), etc.

[0071] Embodiments herein further provide a kit for the identification and differentiation of oral and stomach cancers and grades thereof. The kits include means for determining the presence of at least one cancer protein using the p53 amyloid-binding composition as disclosed in various embodiments herein.

[0072] In one embodiment, the kit includes at least one receptacle for receiving a sample and the p53 amyloid-binding composition as disclosed in various embodiments herein.

[0073] The p53 amyloid-binding composition include at least one p53 specific antibody; at least one agent selected from a group consisting of at leastone amyloid- specific dye and at least one amyloid- specific antibody; wherein the p53 amyloid-binding composition binds with at least one cancer protein.

[0074] Non-limiting examples of p53 specific antibodies include mouse monoclonal anti-human p53 protein DO-1, DO-7 (sc-47698), P53 (DO-7) (MAS- 12557), p53 (FL-393) (sc-6243), Pab 1801 (SC-98), PAb 240 (OP43), BP53-12 (ADI-KAP-PK035-D), and so on.

[0075] Non-limiting examples of amyloid- specific dyes include, fluorescein, cyanine, thioflavin T (Thio T), thioflavin S (Thio S), Congo red, m-L Stilbene, Chrysamine G, 2-(4'-Methylaminophenyl) benzothiazole (BTA-1), PIB, BF-227, X-34, TZDM, FDDNP, MeO-X-04, NIAD 4, Amylo-Glo, Nile red, and combinations thereof and combinations thereof.

[0076] Non-limiting examples of amyloid- specific antibodies include rabbit polyclonal oligomer- specific Al l, amyloid- specific (OC) antibody, anti- mouse-IgG conjugated to Alexa Fluor-488, goat anti-rabbit-IgG conjugated to Alexa Fluor-647 and combinations thereof.

[0077] The kits can optionally include additional components useful for performing the methods described herein, including, but not limited to, an instructional material that describes the performance of a method described herein and specific controls / standards.

[0078] In one embodiment, the kit may include an instruction manual for suitable operational parameters in the form of a label or separate insert. For example, the instructions may provide guidelines and protocols for the utilization of the p53 amyloid-binding composition in the detection process. The instruction manual may further provide detailed step-by-step instructions for sample preparation, ensuring the accuracy and reliability of the results.

[0079] In one embodiment, the kit may include certified reference standards for cancer proteins selected from a group consisting of p53, p63, p73, and isoforms thereof (parafilm embedded p53 / p63 / p73 positive tissues as positive controls), serving as benchmarks for result interpretation and validation.

[0080] The invention is further described by reference to the following examples by way of illustration only and should not be construed to limit the scope of the embodiments disclosed herein. It will be apparent to those skilled in the artthat many modifications, both to materials and methods, may be practiced without departing from the scope of the claimed embodiments.Experimental studies

[0081] Embodiments herein describe an exemplary method for identifying at least one protein from the p53 protein family using the p53 amyloid-binding composition. The method includes collecting freshly frozen samples; fixing and embedding the samples in paraffin to obtain paraffin-embedded samples; cutting thin microscopic sections of paraffin-embedded samples; deparaffinizing the sections with 100% xylene, followed by xylene and ethanol in a 1: 1 ratio; rehydrating the sections in decreasing concentrations of ethanol from 100% to 50%, and finally washing with distilled water; incubating the sections for 2 min with TrypLE express enzyme at 37°C for antigen retrieval; washing the sections with Tris-buffered saline with 0.1% Tween-20, pH 7.4 (TBST), then incubating with 0.2% Triton X-100 in TBST for 10 min and blocking with 2% BSA in TBST; incubating the sections overnight at 4 degrees Celsius with primary antibodies such as mouse monoclonal anti-human p53 protein DO-1, anti -human pab240, rabbit polyclonal oligomer- specific Al l, and amyloid- specific (OC) antibody for immunostaining; washing the sections with TBST followed by incubation at room temperature for 2 h with the secondary antibody such as anti-mouse-IgG conjugated to Alexa Fluor-488, and goat anti-rabbit-IgG conjugated to Alexa Fluor-647; staining the sections with filtered 0.6% thioflavin S (Thio S) for 2 min after immunostaining with an anti-p53 primary antibody and subsequent incubation with an Alexa Fluor-555-conjugated anti-mouse-IgG secondary antibody; washing the sections twice with initially 50% ethanol and then with TBST buffer; mounting the sections using 1% l,4-diazabicyclo-[2.2.2] octane prepared in 90% glycerol and 10% phosphate-buffered saline (PBS) and left for drying; and imaging using Zeiss Axio Observer Z1 inverted confocal fluorescence microscope.

[0082] A set of experiments was performed with the p53 amyloid-binding composition, as disclosed in various embodiments herein, to identify and differentiate histopathological grades of oral and stomach cancer.Experiment 1: Immunohistochemistry of Oral (Grade I, II, III, and IV) and Stomach (Grade II, and III) cancer with anti-p53 and amyloid fibrilspecific OC antibodies

[0083] To examine the p53 status and its accumulation into amyloids in cancer tissues, a double immunofluorescence colocalization study was performed using the amyloid- specific antibody OC and a p53-specific antibody (DO-1, Santacruz Biotechnology, Dallas, TX, USA). The data suggest a high colocalization of p53 with amyloid (OC signal) in all of the cancer tissues of oral (FIGs. 1A, IB, and 1C) and stomach cancers (FIG. 2). In the 59 biopsies (53 tumor tissues and six non-cancerous tissues), it was found that >90% of both oral and stomach tumor tissues were positive for p53 in the amyloid state.

[0084] Interestingly, when the stomach and cancer tissues were analyzed grade-wise, the p53 amyloid content (OC antibody staining) was significantly higher in samples of higher tumor grade for both oral (FIG. 3A) and stomach cancers (FIG. 3B). Similar observations were also seen when fluorescence colocalization studies were performed with p53 antibody and amyloid- specific dye ThioS staining with selected cancer tissues (FIG. 4).

[0085] When a double immunofluorescence study was performed using the Al l antibody (red) and p53-specific antibody (green), the lower-grade cancer tissue of both oral and stomach origin showed a high degree of p53 colocalization with oligomer- specific antibody (Al l), whereas the higher-grade tissues showed negligible colocalization. This suggested that p53 oligomers might be formed at the initial stage of cancer, the levels that subsequently go down in the tissues of higher grades (FIG. 5).Experiment 2: Immunohistochemistry of Oral (Grade I, II, III, and IV) and Stomach (Grade II and III) cancer with anti-p53 and anti-p63 and anti-p53 and anti-p73 antibodies.

[0086] To examine the sequestration of p63 and p73 in p53 amyloids in oral and stomach cancer tissues, immunofluorescence experiments were performed using p53 with p73 or p63 antibodies. 12 oral and six stomach cancer samples were analyzed (three individuals from each cancer grade). p53 was observed to colocalize with p73 (FIG. 6A) and p63 (FIG. 6B) in all the cancer grades for both oral and stomach cancer biopsies. The ImageJ analysis suggestedthat the percentage of colocalization was significantly greater in the higher cancer grades for both oral (FIGs. 9A and 9B) and stomach cancers. In oral cancer, the colocalization of p53 and p73 was ~30% in grade I, which increased up to 70% in grade IV, suggesting that p53 and p73 colocalization is highly correlated with oral cancer grade (FIG. 7A). Similar observations were also seen between stomach grade II (20% colocalization) and grade III (60% colocalization) (FIG. 7B). Similar to p53 and p73 colocalization, we also observed a higher degree of colocalization of p53 and p63 in higher cancer grades for both oral (FIG. 8 A) and stomach cancer tissue (FIG. 8B). The colocalization of p53 and p63 was ~20-25% in oral and stomach grade I tissues, which increased to ~60% for both oral grade IV and stomach grade III tissues.Experiment 3: Separation of soluble and insoluble fractions of tissue lysate, followed by western blot to confirm the presence of p53 in the insoluble fraction

[0087] To directly confirm the p53-p73 and p53-p63 co-aggregation, p53 was immunoprecipitated using anti-p53 antibody from oral biopsies of grade I (P4), III (P27)) and stomach biopsies of grade II (P46), III (P47) followed by western blot analysis with anti-p73 and p63 antibodies. The western blot signal confirmed the presence of p73 and p63 isoforms along with immunoprecipitated p53 in higher- grade cancer samples (grade III) for both oral and stomach tissues (FIGs. 10A, 10B, 10C and 10D). In both the cancer types, p53 was observed to be accumulated in the insoluble fraction, while negligible p53 expression was seen in the normal tissues (FIG. 10A, 10B, 10C, and 10D). Furthermore, to examine the co-immunoprecipitation of p53 and p63, as well as p53 and p73, immunoprecipitation was also carried out using antibodies of p63 and p73. Subsequently, western blot was performed using anti-p53 antibody (DO-1). From the analysis, the presence of p53, along with both p63 and p73, was observed (FIGs. 11A and 11B). This suggests that p63 and p73 coaggregate with p53. The amount of p73 / p63 isoforms was higher in the higher cancer grade than in the corresponding lower cancer grade.Experiment 4: Dot-blot analysis of tissue lysate of Oral (Grade I, II, III, and IV) and Stomach (Grade II, and III) cancer with anti-p53 and amyloid fibril specific OC antibody

[0088] Tissue lysate was quantified using Bradford, and an equal volume of the tissue lysate was spotted on the nitrocellulose membrane. The p53 amyloid load was observed to increase significantly with an increase in the cancer grade for both cancer types. A higher amount of p53 amyloids in higher cancer grades was also consistent with dot-blot analysis (FIGs. 12A, 12B, and 12C) from lysate isolated from various grades of cancer tissue extracts.Experiment 5: Isolation of Tissue amyloid fraction from Oral (Grade IV) and Stomach (Grade III) cancer tissues and subsequent Immuno-TEM imaging with anti-p53 antibody.

[0089] The total amyloid pool was further isolated from representative oral and stomach cancer tissues, which showed a fibril-like morphology when observed under the electron microscope (EM) (FIG. 13 A). Immunoelectron microscopy (immunoEM) using p53 antibody (primary) and 10 nm colloidal gold- conjugated secondary antibody confirmed p53 amyloid, as gold particles were aligned along the length of isolated fibrils from oral and stomach cancer (FIG. 13 A). We further confirmed this using double immunoelectron microscopy of oral cancer tissue (grade III) with an anti-p53 antibody (DOI) and anti-p63 antibody. We found both species in single fibrils (FIG. 13B).Experiment 6: FTIR image analysis of Oral (Grade I, II, III, and IV) and Stomach (Grade II and III) cancer tissues for the quantification of percentage P-sheet structure (amyloid)

[0090] To characterize the amyloid content of the various cancer tissues, we performed Fourier-transform infrared (FTIR) imaging. The FTIR imaging with snap-frozen tumor biopsies showed a higher amount of P-sheet content in higher- grade oral cancers compared to the corresponding lower-grade and non-cancer tissues (FIG. 14A). Similar observations were also obtained for stomach cancer biopsies (FIG. 14B). It is important to note that the higher P-sheet content could be due to other protein amyloids along with p53 amyloids. However, a combined study of immunohistochemistry and label-free FTIR imaging on the identical tissue section (used adjacent sections for both the study) supports that the presence of p53 amyloid might be mostly responsible for higher P-sheet-rich structure in these tissue sections. An FTIR study of these p53 fibrils showed the presence of intense peaks at ~1627 for oral (FIG. 15A) and ~ 1632 for stomach cancer (FIG.15B) in the amide I region, suggesting there is a P-sheet-rich amyloid structure in these p53 aggregates. Interestingly, the P-sheet content (1640-1620 cm1) is absent in oral tumor tissue grade I, but the quantity is enhanced gradually for the oral tumor tissue grade II, III, and IV. A similar trend was observed for the stomach tumor tissue grade II and III, where stomach tumor tissue grade III possessed significantly higher P-sheet content than the stomach tumor tissue grade II.

[0091] The foregoing description of the specific embodiments will so fully reveal the general nature of the embodiments herein that others can, by applying current knowledge, readily modify and / or adapt for various applications such specific embodiments without departing from the generic concept, and, therefore, such adaptations and modifications should and are intended to be comprehended within the meaning and range of equivalents of the disclosed embodiments. It is to be understood that the phraseology or terminology employed herein is for the purpose of description and not of limitation. Therefore, while the embodiments herein have been described in terms of embodiments and examples, those skilled in the art will recognize that the embodiments and examples disclosed herein can be practiced with modification within the scope of the embodiments as described herein.

Claims

CLAIMSWe claim:

1. A p53 amyloid-binding composition for identification of cancer protein aggregates or co-aggregates, comprising: at least one p53 specific antibody; and at least one agent selected from a group consisting of at least one amyloid- specific dye and at least one amyloid- specific antibody, wherein the p53 amyloid-binding composition binds with at least one cancer protein associated with oral and stomach cancers and grades thereof.

2. The composition as claimed in claim 1, wherein the at least one p53 specific antibody is selected from a group consisting of mouse monoclonal anti-human p53 protein DO-1, DO-7 (sc-47698), P53 (DO-7) (MA5- 12557), p53 (FL-393) (sc- 6243), Pab 1801 (SC-98), PAb 240 (OP43), and BP53-12 (ADI-KAP-PK035-D).

3. The composition as claimed in claim 2, wherein the at least one p53 specific antibody is mouse monoclonal anti-human p53 protein DO-1.

4. The composition as claimed in any one of claims 1 to 3, wherein the at least one amyloid-specific dye is selected from a group consisting of fluorescein, cyanine, thioflavin T (Thio T), thioflavin S (Thio S), Congo red, m-I-Stilbene, Chrysamine G, 2-(4'-Methylaminophenyl) benzothiazole (BTA-1), PIB, BF-227, X-34, TZDM, FDDNP, MeO-X-04, NIAD 4, Amylo-Glo, Nile red, and combinations thereof.

5. The composition as claimed in claim 4, wherein the at least one amyloidspecific dye is thioflavin S (Thio S).

6. The composition as claimed in any one of claims 1 to 5, wherein the at least one amyloid-specific antibody is selected from a group consisting of rabbit polyclonal oligomer- specific Al l, amyloid- specific (OC) antibody, anti-mouse-IgG conjugated to Alexa Fluor-488, goat anti-rabbit-IgG conjugated to Alexa Fluor- 647 and combinations thereof.

7. The composition as claimed in claim 6, wherein the at least one amyloidspecific antibody is amyloid- specific (OC) antibody or rabbit polyclonal oligomerspecific Al l.

8. The composition as claimed in any one of claims 1 to 7, wherein the cancer protein is selected from a group consisting of p53, p63, p73, and isoforms thereof.

9. The composition as claimed in any one of claims 1 to 8, wherein the cancer protein further comprises of monomeric, oligomeric, amyloid structure, fibrillar form, aggregate forms, co-aggregate forms, folded, unfolded, and misfolded forms of p53, p63, p73, and isoforms thereof.

10. A method for identification of the cancer protein aggregates or coaggregates, the method comprising: obtaining at least one tissue sample from an individual; contacting the sample with the p53 amyloid-binding composition as claimed in claim 1; and detecting the degree of colocalization of the composition with at least one cancer protein, wherein the cancer protein is selected from a group consisting of p53, p63, p73, and isoforms thereof.

11. The method as claimed in claim 10, wherein the cancer protein further comprises of monomeric, oligomeric, amyloid structure, fibrillar form, aggregate forms, co-aggregate forms, folded, unfolded, and misfolded forms of p53, p63, p73, and isoforms thereof.

12. The method as claimed in claim 10, wherein the p53 amyloid -binding composition comprises at least one p53 specific antibody; and at least one agent selected from a group consisting of at least one amyloid- specific dye and at least one amyloid- specific antibody.

13. The method as claimed in claim 12, wherein the at least one p53 specific antibody is selected from a group consisting of mouse monoclonal anti-human p53 protein DO-1, DO-7 (sc-47698), P53 (DO-7) (MA5- 12557), p53 (FL-393) (sc- 6243), Pab 1801 (SC-98), PAb 240 (OP43), and BP53-12 (ADI-KAP-PK035-D).

14. The method as claimed in claim 13, wherein the at least one p53 specific antibody is mouse monoclonal anti-human p53 protein DO-1.

15. The method as claimed in claim 12, wherein the at least one amyloid-specific dye is selected from a group consisting of fluorescein, cyanine, thioflavin T (Thio T), thioflavin S (Thio S), Congo red, m-I-Stilbene, Chrysamine G, 2-(4'- Methylaminophenyl) benzothiazole (BTA-1), PIB, BF-227, X-34, TZDM, FDDNP, MeO-X-04, NIAD 4, Amylo-Glo, Nile red, and combinations thereof.

16. The method as claimed in claim 15, wherein the at least one amyloid-specific dye is thioflavin S (Thio S).

17. The method as claimed in claim 12, wherein the at least one amyloid-specific antibody is selected from a group consisting of rabbit polyclonal oligomer- specific Al l, amyloid- specific (OC) antibody, anti-mouse-IgG conjugated to Alexa Fluor- 488, goat anti-rabbit-IgG conjugated to Alexa Fluor-647 and combinations thereof.

18. The method as claimed in claim 17, wherein the at least one amyloid-specific antibody is amyloid- specific (OC) antibody or rabbit polyclonal oligomer- specific Al l.

19. A kit for identification and differentiation of oral and stomach cancers and grades thereof, said kit comprising at least one receptacle for receiving a sample; and the p53 amyloid-binding composition as claimed in claim 1.

20. The kit as claimed in claim 19, wherein the p53 amyloid-binding composition comprise of at least one p53 specific antibody; and at least one agent selected from a group consisting of at least one amyloid- specific dye and at least one amyloidspecific antibody.

21. The kit claimed in claim 20, wherein the at least one p53 specific antibody is selected from a group consisting of mouse monoclonal anti -human p53 protein DO-1, DO-7 (sc-47698), P53 (DO-7) (MA5-12557), p53 (FL-393) (sc-6243), Pab 1801 (SC-98), PAb 240 (OP43), and BP53-12 (ADI-KAP-PK035-D).

22. The kit claimed in claim 21, wherein the at least one p53 specific antibody is mouse monoclonal anti-human p53 protein DO-1.

23. The kit claimed in claim 20, wherein the at least one amyloid-specific dye is selected from a group consisting of fluorescein, cyanine, thioflavin T (Thio T), thioflavin S (Thio S), Congo red, m-I-Stilbene, Chrysamine G, 2-(4'- Methylaminophenyl) benzothiazole (BTA-1), PIB, BF-227, X-34, TZDM, FDDNP, MeO-X-04, NIAD 4, Amylo-Glo, Nile red, and combinations thereof.

24. The kit as claimed in claim 23, wherein the at least one amyloid- specific dye is thioflavin S (Thio S).

25. The kit as claimed in claim 20, wherein the at least one amyloid-specific antibody is selected from a group consisting of rabbit polyclonal oligomer- specific Al l, amyloid- specific (OC) antibody, anti-mouse-IgG conjugated to Alexa Fluor- 488, goat anti-rabbit-IgG conjugated to Alexa Fluor-647 and combinations thereof.

26. The kit as claimed in claim 25, wherein the at least one amyloid-specific antibody is amyloid- specific (OC) antibody or rabbit polyclonal oligomer- specific Al l.

27. The kit claimed in claim 19, wherein the kit further comprises an instruction manual and certified reference standards for cancer proteins selected from a group consisting of p53, p63, p73, and isoforms thereof.

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