DESC:F O R M 2

THE PATENTS ACT, 1970 (39 of 1970)
&
THE PATENTS RULES, 2003
COMPLETE SPECIFICATION
[See section 10 and rule 13]

1. TITLE OF THE INVENTION: MOLECULAR MARKER BASED ORAL CYTOLOGY FOR DETECTION OF POTENTIALLY MALIGNANT AND MALIGNANT ORAL LESION

2. APPLICANT (A) Mazumdar Shaw Medical Foundation

(B) Mazumdar Shaw Medical Foundation
A-Block, 8th Floor, Mazumdar Shaw Medical Centre,
#258/A, Narayana Health City, Bommasandra, Bangalore, Karnataka, India, 560099

3. NATIONALITY (C) INDIA

THE FOLLOWING SPECIFICATION PARTICULARLY DESCRIBES THE NATURE OF THIS
INVENTION AND THE MANNER IN WHICH IT IS TO BE PERFORMED
TECHNICAL FIELD OF THE INVENTION
[001] The present invention is in the technical field of molecular marker-based cytology method for the detection/diagnosis of oral potentially malignant (OPML) and malignant lesions. In particular, the invention relates to proteomic profiling, detection and identification of selected proteins that are differentially regulated proteins, in a biological sample from an individual as candidate markers.
BACKGROUND OF THE INVENTION
[002] The age-adjusted incidence rate of Oral Squamous Cell Carcinoma (OSCC) in India is 20 per 100,000 which is one of the highest in the world 1; 80% of OSCC arises from potentially malignant lesions (PML) 2, 3 which have a prevalence of 2.5% to 8.5% 4, 5.
[003] Biopsy and histopathological grading of dysplasia is considered as the benchmark for the management of PML. However, being an invasive procedure in combination with the requirement of a well-trained pathologist for interpretation, biopsies and conventional histology are not feasible as a screening tool in a large volume, high-risk population.
[004] In addition to the challenges of low patient compliance, the invasive nature of the method also negates the possibility of using the method for periodical monitoring essential for disease surveillance.
[005] Brush biopsy and cytology is a non-invasive, relatively simple and inexpensive method for screening of cancer, a method well established in cervical cancer screening programs 6. However, conventional oral cytology has not advanced similarly 7; this may be due to sampling difficulty owing to the different topography of oral cavity and lack of well-defined guidelines for the interpretation of oral cytology subsequently leading to a subjective diagnosis 8.
[006] There have been attempts in recent studies towards addressing these issues, sampling methods has improved due to introduction of new cytology brushes and liquid based cytology techniques9. Current oral cytology using the Hematoxylin/Eosin have less sensitivity (<25%) in detection of oral potentially malignant lesion.
[007] At present, the current oral conventional oral cytology has less sensitivity in detection of oral premalignant lesion and malignant lesion.
[008] Furthermore, there is no molecular probe to differentiate oral premalignant lesion and malignant lesion from benign lesion by cytology.
[009] In addition, there is a need for an improved understanding of the biology of cancer and identification of possible molecular markers that can diagnose and prognosticate epithelial dysplasia.
[010] In addition, currently available methods of oral screening methods include mobile-based imaging, fluorescent based imaging (VeLscope), vital staining (toluidine blue staining), optical imaging (Optical coherence tomography, Confocal microscopy) and oral cytology (Oral CDx). None of the devices have been adapted extensively into clinical practice due to the cost and decreased specificity. Moreover, specificity of these tests reduces when diagnosis of benign lesions are involved. Lack of automation also necessitated the presence of specialist to infer the diagnosis, making it non-feasible in a primary health care setting for early detection/screening.
[011] In summary, there is an urgent need in the art to develop a cytology-based method using molecular markers to improve the diagnostic ability of oral cytology for early detection/screening/diagnosis of oral cancers.
SUMMARY OF THE INVENTION
[012] The present invention provides a marker-based cytology method for the detection/diagnosis of oral potentially malignant (OPML) and malignant lesions by proteomic profiling, detection and identification of selected proteins that are differentially regulated proteins, in a biological sample from an individual as candidate markers. More particularly, the invention provides a set of molecular markers that can detect OPML and malignant lesions with manual liquid-based cytology to distinguish dysplastic, non-dysplastic, squamous cell carcinoma, benign lesions and normal mucosa. In addition, the invention also provides kits that are useful for the practice of the methods of the invention.
[013] In one of the embodiment, the present invention provides a minimally invasive, marker-based cytology method for the detection of oral potentially malignant (OPML) and malignant lesions.
[014] According to a further aspect of the present invention, the two major objectives are,
[015] To determine the most effective biomarkers that can detect OPML and malignant lesions.
[016] To incorporate the most effective biomarker with manual liquid-based cytology to distinguish dysplastic, non-dysplastic, squamous cell carcinoma, benign lesions and normal mucosa.
[017] In preferred embodiments, Objective 1: To determine the most effective biomarkers that can detect OPML and malignant lesions.
[018] Different molecular markers that have potential to distinguish oral dysplastic and neoplastic lesions from benign and normal tissues were selected by detailed literature review.
[019] Phase I: Validation in a smaller cohort of samples (pilot study).
[020] Phase II: Selected markers validated in a large cohort to find best markers.
[021] Objective 2: To incorporate the most effective biomarker/s in aim 1 with manual liquid-based cytology to distinguish benign/dysplastic/squamous cell carcinoma.
[022] In this part of the study, the markers were evaluated for their efficacy in oral cytology; the cytology validation was conducted in two stages; initial validation in cell lines followed by the validation in patient samples.
[023] Objective 3: Oral mucosal nomogram of molecular cytology markers.
[024] Objective 4: Effectiveness of multiplex molecular cytology.
[025] According to a further aspect of the present invention, validation in cell lines were carried out: Head and neck cancer cell lines representing the dysplastic stage (Dysplastic Oral Keratinocytes; DOK) and Oral Squamous Cell Carcinoma (OSCC, CAL 27) were selected and compared to the normal oral epithelial cells from healthy volunteers.
[026] According to aspects of the present invention, Validation in patient samples were carried out: All the selected panel of markers were validated in oral epithelial cells (brush biopsy) collected from patients who are histopathologically diagnosed with benign, High Grade Dysplasia (HGD-Moderate/severe dysplasia and carcinoma-in-situ), Low Grade Dysplasia (LGD- Mild/non-dysplastic) as well as OSCC. The brush biopsy samples from normal mucosa of healthy volunteers were also used as the control.
[027] As will be appreciated by a person skilled in the art the present invention provides a variety of following advantages.
[028] Ability to differentiate OPML and Malignant lesion by cytology from benign lesions, which was not successfully accomplished by other methods.
[029] Inventors have provided a panel of 4 markers CD44, CyclinD1, SNA-1 and MAA that could differentiate OPML and malignant lesion from benign lesion by histology and cytology with more than 80% sensitivity.
[030] Additionally, the system allows for further improvement by making the detection amenable to automated image analysis, a preferred system for the development of a Point of Care system.
[031] In preferred embodiments, the present invention can be used,
[032] To improve the cytology platform for detection of OPML
[033] Early detection tool
[034] Can be developed as Point of care diagnostic tool for detection of OPML and malignant lesion.
[035] In yet another embodiment, preferably, the sample is oral tissue for the evaluation of these markers. However, other samples can be selected from body fluid, wherein said body fluid is urine, peripheral blood, or saliva.
[036] According to a further exemplary aspect of the present invention, non-limiting and example of use of biomarkers in the detection of head and neck cancers including oral cancers, cancers of larynx , pharynx, primary or secondary cancers as a cost-effective method for screening on large scale, for prognosis, screening at various stages of cancers in a subject during and post -treatment, in the form of kits, slides, and such other easily usable, disposable, multi-utility kits for measuring the preferred biomarkers in cytology to detect oral cancers including oral cancers with specificity, sensitivity, accuracy, speed, reliability and reproducibility.
[037] The advantages of this invention include but not limited to,
[038] Individual or combination of markers can be used to develop assay systems for the early diagnosis of high-risk lesions.
[039] Individual or combination of markers can be used for community based screening of high-risk populations to identify patients at risk for developing oral premalignant lesion and/or cancer.
[040] Individual or combination of markers can be used to develop monitoring or surveillance systems to monitor disease progression.
[041] Individual or combination of markers can be used to predict the development of nodal metastasis.
[042] Scoring pipeline to identify the markers of high technical and functional relevance for further validations
[043] The marker panel can be used for the development of a Point-of-Care assay system that can be applied towards early detection, screening, and disease progression.
[044] As will be appreciated by person skilled in the art, the present invention provides following exemplary embodiments / claims,
[045] A method for detecting or diagnosing or predicting oral potentially malignant (OPML) and malignant lesions in a subject having or at risk of developing OPML, the method comprising:
a. obtaining a sample from the subject;
b. identification of selected proteins that are differentially regulated in a sample, wherein the protein is selected from the group comprising of CD44, CyclinD1, SNA-1, MAA, and combinations thereof; and
c. comparing the cytology of the samples; wherein the levels of SNA-1, MAA, CYCLIN D1 and CD44 proteins in the patient sample, when compared to the SNA-1, MAA, CYCLIN D1 and CD44 proteins level in the control sample is/are indicative that the subject has or is at risk of developing OPML.
[046] The method of [045], wherein the four markers were evaluated in cytology by using either HRP conjugated secondary antibodies (CD44, CYCLIN D1) or by FITC conjugation (SNA-1, MAA), wherein the level of SNA-1, MAA and CD44 in the sample is at a higher level compared to the uptake in normal oral epithelial cells.
[047] The method as claimed in [045], wherein the sample in step a) is selected from a group consisting of oral mucosal cells, saliva, blood, and combinations thereof.
[048] The method as claimed in [045], wherein step b) comprises carrying out immunocytochemistry, immunohistochemistry and combinations thereof.
[049] A method for aiding assessment of a patient's likely severity or likelihood of premalignant lesions and cancer, or aiding in selection of a treatment regime for the patient, or aiding in assessment of a treatment regime, or the detection and diagnosis of oral or oral potentially malignant (OPML) or malignant lesions in a subject, the method comprising:
a. determining in a biological sample from the patient the amount of the expression level of at least one protein selected from the group consisting of CD44, CyclinD1, SNA-1, MAA and a combination thereof; and
b. comparing the determined expression levels of said protein in said biological sample with the level in a reference.
[050] The method according to any one of the preceding claims, wherein the cancer is selected from the group consisting of oral potentially malignant (OPML) and Oral cancers.
[051] A method of detecting pre-cancer/cancer in a patient, the method comprising of collecting the cells from the patient and staining the same with an anti- CyclinD1 or anti- CD44 antibodies or with SNA-1 or MAA lectins or anti- CYCLIN D1 antibody or a fragment or derivative thereof labelled with a detectable label, allowing the labelled antibody to locate to the cancer, and imaging the cancer.
[052] The method according to any one of the preceding claims, wherein the detection of oral potentially malignant (OPML) and malignant lesions in cancers of oral cavity (tongue, buccal mucosa, palate, RMT, gingiva) can be detected in a minimally invasive manner, wherein the method can be extended to larynx, pharynx, oropharynx, primary or secondary cancers, apart from oral cancers in animals that includes humans as a cost-effective method for screening on large scale, for prognosis, screening at various stages of cancers in the subjects during and post -treatment, in the form of kits, slides, and such other easily usable, disposable, multi-utility kits containing the preferred biomarkers for detecting preferably head and neck, oral cancers with specificity, sensitivity, accuracy, speed, reliability and reproducibility.
[053] A kit for diagnosing or predicting or likelihood of progression of cancer, or aiding in selection of a cancer treatment regime for the patient, or aiding in assessment of a cancer treatment regime, wherein the kit comprising at least one reagent for determining the amount of the expression level of at least one gene selected from the group comprising of CD44, CyclinD1, SNA-1, MAA and a combination thereof.
[054] The kit as according to [053], wherein the kit comprising of at least one reagent that specifically binds to a cancer biomarker and a package insert containing instructions using the kit, wherein the marker panel can be used for the development of a Point-of-Care assay system that can be applied towards early detection, screening, and disease progression.
[055] In summary, the present invention deals with a minimally invasive, molecular marker-based cytology method for the detection/diagnosis of oral potentially malignant (OPML) and malignant lesions. In particular, the invention relates to proteomic profiling, detection and identification of selected proteins that are differentially regulated proteins, in a biological sample from an individual as candidate markers. More particularly, the invention provides a set of molecular markers that can detect OPML and malignant lesions with manual liquid-based cytology to distinguish dysplastic, non-dysplastic, squamous cell carcinoma, benign lesions and normal mucosa. In addition, the invention also provides kits that are useful for the practice of the methods of the invention.
[056] Several aspects of the invention are described below with reference to examples for illustration. However, one skilled in the relevant art will recognize that the invention can be practiced without one or more of the specific details or with other methods, components, materials and so forth. In other instances, well-known structures, materials, or operations are not shown in detail to avoid obscuring the features of the invention. Furthermore, the features/aspects described can be practiced in various combinations, though only some of the combinations are described herein for conciseness.
BRIEF DESCRIPTION OF THE DRAWINGS
[057] Example embodiments of the present invention will be described with reference to the accompanying drawings briefly described below according to the aspects of present invention.
[058] FIG. 1 Work flow of study: Molecular cytology using Lectin and CD44 can improve the oral cytology which improve sensitivity, specificity and decreased inter-observer variation
[059] FIG. 2 illustrates a schematic representation of workflow of the study, according to the aspects of present invention.
[060] FIG. 3 illustrates the profile of markers (n=11) in the different patient groups: Box and Whisker plot analysis showing distribution of total IHC intensity score of histopathologically confirmed normal oral mucosa (n=10), benign (n=10), HGD (n=10) and OSCC (n=10). SNA-1, WGA and MAA were able to delineate HGD/OSCC from Normal/Benign lesions (P<0.05). Cyclin D1, CD44 and S100A7 were able to delineate HGD from benign lesion. *p Value X < 0.05, XX, as provided in one embodiment.
[061] FIG. 4 illustrates the receiver operating characteristic (ROC) Analysis of the markers A: ROC curves of markers according to IHC scores in differentiating normal & benign lesions from HGD & OSCC. The SNA-1, S100A7, P53, and CD44 expression showed best sensitivity and specificity (>75%) with highest Area under Curve (AUC> .75). B: Graph representing AUC of markers, according to the aspects of present invention.
[062] FIG. 5 illustrates the expression of the validated markers in representative images: Figure representing H/E and IHC images of Normal, Benign, HGD and OSCC of 7 markers (Magnification: 100x), according to the aspects of present invention.
[063] FIG. 6 shows the marker validation, profile of markers (n=7) in large cohort: Box and Whisker plot showing CD44, SNA-1, MAA and CyclinD1 were significantly delineate Normal/Benign lesions from HGD/OSCC (P<0.05). *P Value X < 0.05, XX <0.005, XXX<0.0005, as provided in one embodiment.
[064] FIG. 7 illustrates the ROC Analysis: ROC curves of 7 markers (A) and AUC (B) in differentiating Normal/Benign (n=50) from HGD/OSCC (n=81). ROC curve analysis shows SNA-1, CD44, MAA and CyclinD1 have high AUC >.75, as provided in one embodiment.
[065] FIG. 8 Shows the Marker profile in Cell lines: Box and whisker plot showing the intensity of cell lines and normal subject of CD44 (A), MAA (B) and SNA-1(C). CAL 27, DOK shows significantly high uptake of markers compared to normal oral epithelial cells (p<0.0001). Cyclin D1 didn’t show any uptake, as provided in one embodiment.
[066] FIG. 9 illustrates the SNA-1 expression profile in different cohorts: Box and Whisker plot of percentage of cells (% of cells showing higher expression that in lesion site compared to normal site) (A), maximum intensity (B) and mean intensity score (C) of SNA-1 stained cells of histologically confirmed OSCC, HGD, LGD and Benign cases. The percentage and maximum intensity score of SNA-1 is significantly higher (p<.05) in HGD/OSCC subjects compared to the uptake in LGD/Benign/normal (respective tables below box plot represents ANOVA analysis between cohorts). Logistic regression shows sensitivity and specificity of 83 and 70.8 respectively for combined SNA% and maximum intensity, according to the aspects of present invention.
[067] FIG. 10 illustrates the MAA expression profile: Box and Whisker plot of percentage of cells of higher expression (A), maximum intensity (C) and mean intensity score (B) of MAA stained cells of histologically confirmed OSCC, HGD, LGD and Benign cases. The percentage score of MAA is significantly lower (p<.05) in LGD compared with HGD/OSCC subjects. (respective tables below box plot represents ANOVA analysis between cohorts). The mean intensity is significantly higher in cancer, logistic regression shows sensitivity and specificity of 40.5 and 70.65 respectively.
[068] FIG. 11 shows the CD44 expression profile in different cohorts: Box and Whisker plot of percentage of cells showing higher expression (>4 Intensity) (A), percentage of cells showing nuclear positivity (B) and maximum intensity score (C) of CD44 stained cells. All variables show that HGD/OSCC is significantly higher expression. The maximum intensity and nuclear positivity were selected by logistic regression analysis and showed sensitivity and specificity of 53.3 and 89.5 respectively in delineation of the groups, according to the aspects of present invention.
[069] FIG. 12 illustrates the CyclinD1 expression profile: Box and Whisker plot of percentage of cells showing higher expression (>4 Intensity) (A), maximum intensity score (B) and percentage of cells showing nuclear positivity (C) of MAA stained cells of histologically confirmed OSCC, HGD, LGD and Benign cases. The maximum Intensity and percentage of nuclear positivity delineated OSCC from HGD/LGD/Benign cases (respective tables below box plot represents ANOVA analysis between cohorts). The maximum intensity was selected by logistic regression analysis (stepwise) with sensitivity and specificity of 58.4 and 65.85 respectively, according to the aspects of present invention.
[070] FIG. 13 Shows the representative composite image (combined FITC, TRITC and DAPI) of 3 sites (5 X 5 cells) cropped from actual image, as provided in one embodiment.
[071] FIG. 14 (A and B) illustrates the SNA-1 AND CD44 intensity of 3 sites. Both markers showed that there is no Significant difference (p>0.05) between site, as provided in one embodiment.
[072] FIG. 15 illustrates the depicting comparison of SNA-1 (A) and CD44 (B) expression age specific subjects (<40 years and >/=40 years) CD44 showed Significantly (p<0.05) high expression in >40 age group individual, as provided in one embodiment.
[073] FIG. 16 illustrates the correlation (A) of SNA-1 (red line) and CD44 (black line) expression with age (blue line) in normal individuals. CD44 shows (B) significant correlation with age (r= 0.602, CI= 0.3746-0.7610), as provided in one embodiment.
[074] FIG. 17 illustrates the representing multiplexed epifluorescence composite image (combined FITC, TRITC and DAPI in 200x) inset shows cropped zoomed-in images of CD44 and SNA-1 staining, according to the aspects of present invention.
[075] FIG. 18 illustrates the representative cell images of benign, HGD and OSCC subjects (5x5 cropped cells from original images), according to the aspects of present invention.
[076] FIG. 19 illustrates the SNA-1 expression (maximum Intensity and average intensity) were significantly different among cohorts (table below graph shows ANOVA post hoc analysis), as provided in one embodiment.
[077] FIG. 20 illustrates the CD44 expression: CD44- maximum Intensity and average intensity were significantly different among cohorts (table below graph). The results are from ANOVA analysis, as provided in one embodiment.
[078] FIG. 21 illustrates the ROC curve analysis of markers delineating OSCC/HGD from LGD/Benign/Normal: ROC curve analysis (A-D) showed that SNA-average/maximum intensity had better sensitivity/specificity. SNA maximum Intensity (B) delineated OSCC/HGD from LGD/benign with high sensitivity (92.9%) and specificity (82.4%). Comparison of ROC (E) curve analysis showed that SNAmax has high AUC (0.90). Logistic regression (stepwise method, F) selected SNA/CD44 average as best variables to delineate OSCC/HGD from LGD/Benign/normal with more than 80% sensitivity, according to the aspects of present invention.
[079] FIG. 22 illustrates the Sequential analysis to delineate OSCC and HGD from others: Comparison of ROC curve analysis showed that SNA average intensity (A) delineated OSCC from others with a sensitivity of 100% (AUC=0.95) and SNA max intensity (B) delineate HGD from others by more than 82% sensitivity and specificity (AUC=0.82), according to the aspects of present invention.
[080] In the drawings, like reference numbers generally indicate identical, functionally similar, and/or structurally similar elements. The drawing in which an element first appears is indicated by the leftmost digit(s) in the corresponding reference number.
DETAILED DESCRIPTION OF THE INVENTION
[081] It is to be understood that the present disclosure is not limited in its application to the details of construction and the arrangement of components set forth in the following description or illustrated in the drawings. The present disclosure is capable of other embodiments and of being practiced or of being carried out in various ways. Also, it is to be understood that the phraseology and terminology used herein is for the purpose of description and should not be regarded as limiting.
[082] The use of “including”, “comprising” or “having” and variations thereof herein is meant to encompass the items listed thereafter and equivalents thereof as well as additional items. The terms “a” and “an” herein do not denote a limitation of quantity, but rather denote the presence of at least one of the referenced item. Further, the use of terms “first”, “second”, and “third”, and the like, herein do not denote any order, quantity, or importance, but rather are used to distinguish one element from another.
[083] As used herein, the singular forms “a”, “an”, and “the” include both singular and plural referents unless the context clearly dictates otherwise. By way of example, “a dosage” refers to one or more than one dosage.
[084] The terms “comprising”, “comprises” and “comprised of” as used herein are synonymous with “including”, “includes” or “containing”, “contains”, and are inclusive or open-ended and do not exclude additional, non-recited members, elements or method steps.
[085] All documents cited in the present specification are hereby incorporated by reference in their totality. In particular, the teachings of all documents herein specifically referred to are incorporated by reference.
[086] Example embodiments of the present invention are described with reference to the accompanying figures.
[087] In the drawings, like reference numbers generally indicate identical, functionally similar, and/or structurally similar elements. The drawing in which an element first appears is indicated by the leftmost digit(s) in the corresponding reference number.
[088] Definitions
[089] The following terms are used as defined below throughout this application unless otherwise indicated.
[090] The terms “tumour” or “tumour tissue” refer to an abnormal mass of tissue which results from uncontrolled cell division. A tumour or tumour tissue comprises “tumour cells” which are neoplastic cells with anomalous growth properties and no functional bodily function. Tumours, tumour cells and tumour tissue can be benign or malignant.
[091] "Marker" or "biomarker" are used interchangeably, and in the context of the present invention refer to a polypeptide, which is differentially present in a sample collected from patients having HNSCC as compared to a comparable sample taken from control subjects.
[092] The phrase "differentially present" refers to differences in the quantity of the marker present in a sample taken from patients as compared to a control subject. A biomarker can be differentially present in terms of frequency, quantity or both.
[093] "Diagnostic" means identifying a pathologic condition.
[094] The terms "detection", "detecting" and the like, may be used in the context of detecting markers or biomarkers.
[095] A "test amount" of a marker refers to an amount of a marker present in a sample being tested. A test amount can be either in absolute amount (e.g., µg/ml) or a relative amount (e.g., relative intensity of signals).
[096] The terms "polypeptide," "peptide" and "protein" are used interchangeably herein to refer to a polymer of amino acid residues. "Polypeptide," "peptide" and "protein” can be modified, e.g., by the addition of carbohydrate residues to form glycoproteins.
[097] "Detectable moiety" or a "label" refers to spectroscopic, photochemical, biochemical, immunochemical, or chemical means of detection of a composition. For example, labels may include 32P, 35S, fluorescent dyes, biotin-streptavidin, dioxigenin, haptens, electron-dense reagents, and enzymes. The detectable moiety generates a measurable signal that can quantify the amount of bound detectable moiety in a sample. Quantitation of the signal is done by scintillation counting, densitometry, or flow cytometry.
[098] "Antibody” refers to a polypeptide ligand encoded by an immunoglobulin gene(s), which specifically binds and recognizes an epitope.
[099] The terms "subject", "patient" or "individual" generally refer to a human or mammals.
[0100] "Sample" refers to a polynucleotides, antibodies fragments, polypeptides, peptides, genomic DNA, RNA, or cDNA, polypeptides, a cell, a tissue, and derivatives thereof may comprise a bodily fluid or a soluble cell preparation, or culture media, a chromosome, an organelle, or membrane isolated or extracted from a cell.
[0101] Subject refers to a subject or patient can include, but is not limited to, mammals such as bovine, avian, ovine, porcine, canine, equine, feline, or primate animals (including humans and non-human primates).
[0102] The subject can have a pre-existing disease or condition, such as cancer. Furthermore, the subject may not have any known pre-existing condition. In addition, the subject may also be non-responsive to an existing or past treatment, such as a treatment for cancer.
[0103] “Body fluid” refers to, but is not limited to, plasma, serum, urine, peripheral blood, sera, plasma, ascites, urine, cerebrospinal fluid (CSF), sputum, saliva, bone marrow, synovial fluid, aqueous humor, amniotic fluid, cerumen, breast milk, broncheoalveolar lavage fluid, semen, prostatic fluid, cowper's fluid or pre-ejaculatory fluid, female ejaculate, sweat, fecal matter, hair, tears, cyst fluid, pleural and peritoneal fluid, pericardial fluid, lymph, chyme, chyle, bile, interstitial fluid, menses, pus, sebum, vomit, vaginal secretions, mucosal secretion, stool water, pancreatic juice, lavage fluids from sinus cavities, bronchopulmonary aspirates, blastocyl cavity fluid, or umbilical cord blood.
[0104] The term “oral cancer” refers to a group of malignant or neoplastic cancers originating in the oral cavity of an individual. Non-limiting examples of oral cancers include cancers of the buccal vestibule, hard or soft palate, tongue, gums (including gingival and alveolar carcinomas), lingual cancer, buccal mucosa carcinoma, and the like.
[0105] The oral cavity includes the buccal mucosa, upper and lower alveolar ridges, floor of the mouth, retromolar trigone, hard palate, and anterior two thirds of the tongue.
[0106] “Periodontal disease” refers diseases affecting the gums of an individual, including gingivitis, periodontitis, and the like.
[0107] “Therapeutically effective amount or dose” refers to a dose that produces effects for which it is administered. The exact dose depends on the purpose of the treatment.
[0108] Metastasis” refers to spread of a cancer from the primary origin to other tissues and parts of the body, such as the lymph nodes.
[0109] “Prognosis” refers to prediction of the likelihood of metastasis, predictions of disease free and overall survival, the probable course and outcome of cancer therapy, or the likelihood of recovery from the cancer, in a subject.
[0110] “Diagnosis” refers to identification of a disease state, such as cancer in a subject. The methods of diagnosis provided by the present invention can be combined with other methods of diagnosis well known in the art. Non-limiting examples of other methods of diagnosis include detection of known disease biomarkers in pral tissue samples, co-axial tomography (CAT) scans, positron emission tomography (PET), oral radiography, oral biopsy, radionuclide scanning, and the like.
[0111] “Nucleic acid” refers to deoxyribonucleotides or ribonucleotides and polymers thereof in either single- or double-stranded form, and complements thereof.
[0112] The terms “CD44” refers to a cell surface adhesion receptor that is highly expressed in many cancers and regulates metastasis, “CyclinD1” refers to a regulatory subunit of cyclin-dependent kinases, “SNA-1” refers to lectin Sambucus nigra (SNA1), and “MAA” refers to lectin Maackia amurensis.
[0113] A particular nucleic acid sequence may also implicitly encompass conservatively modified variants thereof (e.g., degenerate codon substitutions) and complementary sequences, in addition to the sequence explicitly indicated. Furthermore, degenerate codon substitutions may be achieved by generating sequences in which the third position of one or more selected (or all) codons is substituted with mixed-base and/or deoxyinosine residues.
[0114] The cancer characterized by the methods of the invention can comprise, without limitation, a carcinoma, a germ cell tumor, a blastoma, a sarcoma, a lymphoma or leukemia, or other cancers.
[0115] Diagnostic and Prognostic Methods
[0116] Inventors addressed the needs of developing a minimally invasive, marker-based cytology method for the detection of oral potentially malignant (OPML) and malignant lesions. The two objectives are
[0117] Work flow of the study is mentioned in FIG 2.
[0118] To determine the most effective biomarkers that can detect OPML and malignant lesions
[0119] To incorporate the most effective biomarker with manual liquid-based cytology to distinguish dysplastic, non-dysplastic, squamous cell carcinoma, benign lesions and normal mucosa
[0120] STRATEGY EMPLOYED AND EXAMPLE EMBODIMENTS OF THE PRESENT INVENTION,
[0121] Objective 1: To determine the most effective biomarkers that can detect OPML and malignant lesions
[0122] Different molecular markers that were demonstrated to have potential to distinguish oral dysplastic and neoplastic lesions from benign and normal tissues were selected by detailed literature review.
[0123] Phase I: Validation in a smaller cohort of samples (pilot study).
[0124] Phase II: Selected markers validated in a large cohort to find best markers.
[0125] Objective 2: To incorporate the most effective biomarker/s in aim 1 with manual liquid-based cytology to distinguish benign/dysplastic/squamous cell carcinoma.
[0126] In this part of the study, the markers were evaluated for their efficacy in oral cytology; the cytology validation was conducted in two stages; initial validation in cell lines followed by the validation in patient samples.
[0127] Validation in cell lines: Head and neck cancer cell lines representing the dysplastic stage (Dysplastic Oral Keratinocytes; DOK) and OSCC (CAL 27) were selected and compared to the normal oral epithelial cells from healthy volunteers.
[0128] Validation in patient samples: All the selected panel of markers were validated in oral epithelial cells (brush biopsy) collected from patients who are histopathologically diagnosed with benign, High Grade Dysplasia (HGD-Moderate/severe dysplasia and carcinoma-in-situ), Low Grade Dysplasia (LGD- Mild/non-dysplastic) as well as OSCC. The brush biopsy samples from normal mucosa of healthy volunteers were also used as the control.
[0129] Objective 3: Oral mucosal nomogram of molecular cytology markers The multiplex staining of the selected markers (n=2) was initially standardized. Their expression was assessed in epithelial cells of oral cavity collected from healthy individuals without any habits/oral lesions (n=15, 45 sites).
[0130] Objective 4: Effectiveness of multiplex molecular cytology Multiplex molecular cytology was performed in OSCC (n=25)/OPML (n=24)/ Benign(n=17) patients. The analysis was carried out using Image J.
[0131] Results:
[0132] Objective 1:
[0133] The panel of markers selected for this study by literature survey included those specifying cell division, tumour suppressors, transcriptional regulation, stem cell markers (n=8) and aberrant glycosylation (lectin; n=3) markers. (Table 1). These markers were validated by IHC validation in two phases.
[0134]
[0135] Table 1: Molecular markers selected for Immunohistochemistry, Immunocytology and Immunofluorescence experiment.
[0136] Phase I (Pilot study): The markers (n=11) were initially validated in a cohort of ten patients from each group (normal, benign, HGD and OSCC) by IHC as well as lectin histology and the expression scored as per the standard protocols. The expression patterns were compared between the cohorts (FIG 3) and ROC curve analysis (FIG 4) was done. The markers were selected based on the presence of any one criteria mentioned below
[0137] Differentiate HGD from Normal/benign (p<0.05),
[0138] ROC curve analysis- AUC >0.75 and
[0139] Sensitivity/specificity>75% to differentiate HGD/malignant lesions from Normal/Benign oral lesions.
[0140] Phase II (Large cohort Validation): The seven markers selected by pilot study were validated in a large patient cohort (n=131), which included the normal (n=20), benign (n=30), HGD (n=40) and malignant (n=41). The markers were profiled by IHC/lectin histology (FIG 5). The markers were selected based on the presence of all criteria mentioned above. The Box and whisker plot and ROC curve analysis of all markers shown in FIG 6 & 7.
[0141] Objective 2:
[0142] In the next part of the study, the markers were evaluated for their efficacy in oral cytology; the cytology validation was conducted in two stages i) validation in cell lines and ii) validation in patient samples.
[0143] Validation in cell lines. The four markers were selected from objective 1 were evaluated in cytology by using either HRP conjugated secondary antibodies (CD44, CYCLIN D1) or by FITC conjugation (SNA-1, MAA). Image analysis (using Image J) revealed that the uptake of SNA-1, MAA and CD44 were significantly higher (p=0.0001) in CAL 27 and DOK cell lines compared to the uptake in normal oral epithelial cells (FIG 8).
[0144] SNA-1/MAA: Comparison of the percentage of cells (% of cells with higher intensity compared to their respective normal site) (FIG 9A), mean intensity (FIG 9B) and maximum recorded intensity (FIG 9C) across the cohorts indicated that the percentage of cells and maximum intensity score of SNA-1 is significantly higher (p<.05) in HGD/OSCC subjects compared to LGD/benign (ANOVA). HGD/OSCC were differentiated with 83.5% sensitivity and 70.8% specificity (Fig 9D logistic regression; AUC: 0.81; p<0.0001). Similar analysis indicated that the mean intensity score (FIG 10 A, B &C) of MAA is significantly higher (p<.05) in OSCC compared to benign/LGD/HGD and delineated OSCC/HGD with sensitivity of (Fig 10D) 41% and specificity of 89%.
[0145] CD44/Cyclin D1: Expression pattern was evaluated by percentage of cells having >4 intensity score (FIG 11A), overall nuclear expression pattern (+/-), percentage of cells showing nuclear positivity (FIG 11B) and the maximum intensity score in each patient (FIG 11C). Logistic regression showed that nuclear positivity and maximum intensity of CD44 has a high specificity (89.5%) but low sensitivity (53.3%) in delineating OSCC/HGD (Fig 11D; AUC: 71%). The median maximum intensity showed a progressive increase (OSCC: 5; HGD: 5; LGD: 4; benign: 4) (p<0.05, ANOVA). Similarly, logistic regression indicated that maximum intensity of CyclinD1 had specificity and sensitivity of 65.85 and 58.44 in delineating OSCC/HGD (AUC: 63%) (FIG 12A-D).
[0146] Combination of all markers: Multivariate logistic regression analysis performed to assess the efficacy of the combination (Table 2) (14 variables; MedCalc14.8.1; stepwise method), selected 4 best variables, CD44/CycinD1 nuclear positivity, SNA-1 maximum intensity and SNA-1 percentage score (Sensitivity: 93.33%; Specificity: 72%) in differentiating OSCC/HGD (p<0.0001, AUC= 0.901). Based on these results, we selected CD44 and SNA-1 for multiplex validation and owing to the low odds ratio of CyclinD1 (Table 2) and the lack of capability to delineate HGD.
Coefficients and Standard Errors
Variable Coefficient Std. Error P
CD44N +ve 3.20722 0.78131 <0.0001
CyD1N +ve -1.84117 0.67125 0.0061
SNA-1 % 0.040726 0.013173 0.002
SNA-1 MAX 0.074283 0.023528 0.0016
Constant -3.9182
Odds Ratios and 95% Confidence Intervals
Variable Odds ratio 95% CI
CD44N_ 24.7103 5.3433 to 114.2736
CyD1N_ 0.1586 0.0426 to 0.5913
SNA1% 1.0416 1.0150 to 1.0688
SNA1MAX 1.0771 1.0286 to 1.1279
Hosmer & Lemeshow test
Chi-squared 9.1375
DF 8
Significance level P = 0.3308
Classification table (cut-off value p=0.5)
Actual group Predicted group Percent correct
0 1
Y = 0 28 11 71.79%
Y = 1 5 70 93.33%
Percent of cases correctly classified 85.96%
ROC curve analysis
Area under the ROC curve (AUC) 0.901
Standard Error 0.0318
95% Confidence interval 0.830 to 0.949
[0147] Table 2: Multivariate Logistic regression analysis shows the combination of 3 markers using 14 variables, CD44/CycinD1 Nuclear positivity and SNA Maximum intensity score, have sensitivity and specificity of 93.3% and 72% respectively in differentiating OSCC/HGD from LGD/Benign subject.
[0148] Objective 3: Oral mucosal nomogram of molecular cytology markers
[0149] SNA1-CD44 Nomogram: Multiplexing experiment was standardized and assay carried out for normal cells collected from the different sites (buccal mucosa, tongue-lateral, gingiva) of oral cavity from healthy volunteers (n=15, sites: 3) without risk habits. The age range of the subjects was 24-56 years with a male to female ratio of 2:3. Multiplexed immuno-cytological (FIG 13) analysis was carried out in these samples with SNA1 and CD44 and the baseline intensity values calculated with regard to site-specific (FIG 14A-B) and age-specific (FIG 15A-B) changes. CD44 showed significantly high expression in above 40 years of individual and significant correlation with age (FIG 16A-B), while SNA-1 showed no significant difference age/site wise.
[0150] Objective 4: Efficacy of multiplexed marker profiling in molecular cytology
[0151] Oral cytology with SNA1-CD44 multiplex assay: The multiplexed marker profiling (FIG 17 & 18) with SNA-1-TRITC and CD44-FITC in OSCC/OPML/benign patients indicated that SNA-1 and CD44 expression (FIG 19 & 20) were significantly different among cohorts (p<0.05). ROC curve analysis (FIG 21) showed that SNA maximum intensity delineated OSCC/HGD from LGD/benign/normal with high sensitivity (92.9%) and specificity (82.4%). Logistic regression (SNA maximum and average intensity, CD44 maximum and average intensity) by stepwise method selected SNA average and CD44 average as the best variables with sensitivity and specificity of 81% and 88.2% respectively (FIG 21F, AUC =0.91). Additionally, SNA-average intensity also delineated OSCC from others with high sensitivity (100%) and specificity (82.35%) (FIG 22B, AUC=0.95), and SNA-max intensity alone delineated HGD from LGD/benign with a sensitivity/specificity of 82.4% (FIG 23C, AUC=0.81).
[0152] Multivariate Linear Regression model for interaction between CD44-SNA1 (Table 3): Linear regression model was developed with the variables age, site and marker expression (CD44 average and maximum Intensity, SNA average and maximum intensity) as independent variables and histology diagnosis (ordinal values) as dependent variable (LGD/Benign/normal=1, HGD=2 and OSCC=3). Model shows significant interaction between aberrant glycoprotein (CD44) and aberrant glycosylation (SNA-1); Regression model was developed with 5 predictors; site and marker expressions, with R2=0.74 (adjusted R2=0.70; p=7.1e-22).
Table 3 Linear regression model with interaction shows significant interaction between glycoprotein (CD44) and aberrant glycosylation (SNA-1)
[0153] Manual cytology by pathologist: Convention cytology-based diagnosis was also carried out from all the subjects in the final validation set (n=122). The oral pathologist diagnosed the slides as atypia positive/negative; a sensitivity and specificity of 80% (20/25) and 76% (61/80) respectively was observed for delineating OSCC from LGD/benign/normal subjects, while HGD delineation could be carried out with 23.5% sensitivity and 76% specificity (Table 4).
[0154] Final Outcome of study
[0155] The study focused on developing molecular markers for improving the cytology method of delineation of OSCC and HGD lesions from LGD/benign/normal lesions. Immunohistochemical analysis followed by cytology-based validation identified CD44 and SNA-1 as the best combination, which delineated OSCC with 100% sensitivity and HGD with 82% sensitivity. Nomogram of the marker combination was also established. In addition, the CNN-based image processing algorithm developed was capable of delineating OSCC/HGD with more than 80% sensitivity and specificity (Table 4), which is instrumental in translating the findings of this study into a PoC system. Additionally, the two markers identified, a glycoprotein (CD44) and the lectin detecting aberrant glycosylation (SNA-1) showed a correlation; experimental validation will indicate the relevance of this finding in early oral cancer.
[0156]
[0157] Table 4 Sensitivity and specificity of Molecular cytology: depicting sensitivity and specificity of conventional cytology, molecular cytology and CNN assisted molecular analysis in risk stratifying patients in OSCC and HGD.
[0158] According to a non limiting exemplary aspect of the present invention, the primary advantage of the current invention is the non-invasive mode of diagnosis/prognosis. The biomarkers identified in the study are predicted to identify dysplastic lesions. Additionally, the use of these markers will increase the accuracy of the diagnosis since these changes are highly specific.
[0159] According to a non limiting exemplary aspect of the present invention, these markers also have the probability of being used to assess the progression/regression of oral cancer after treatment. Since the molecular changes precede the histological and clinical changes, the use of this method can also facilitate early detection/prognosis.
[0160] Compositions, Kits and Integrated Systems; the invention provides compositions, kits and integrated systems for practicing the assays/methods described herein using antibodies specific for polypeptides and/or lectins etc.The kits typically include a probe that comprises an antibody-lectin that specifically binds to a specific polypeptides or carbohydrate moieties in the protein, and a label for detecting the presence of the probe. In addition, the kits may include several antibodies specific for, or lectin specific to the protein/carbohydrate moieties of the invention.
[0161] Major advantage of the current invention is non-invasive method of detection.
[0162] The major advantages/improvements of our system are
[0163] Ability to differentiate OPML and Malignant lesion by cytology from benign lesions, which was not successfully accomplished by other methods.
[0164] We have provided a panel of 4 markers CD44, CyclinD1, SNA-1 and MAA that could differentiate OPML and malignant lesion from benign lesion by histology and cytology with more than 80% sensitivity.
[0165] Additionally, the system allows for further improvement by making amenable to automated image analysis, a preferred system for the development of a Point of Care system.
[0166] The possible uses of this invention include but not limited to,
[0167] Improve the cytology platform for detection of OPML, early detection tool.
[0168] Individual or combination of markers can be used to develop assay systems for the early diagnosis of high risk lesions, community-based screening of high-risk populations to identify patients at risk for developing oral premalignant lesion and/or cancer, and to develop monitoring or surveillance systems to monitor disease progression.
[0169] The marker panel can be used for the development of a Point-of-Care assay system that can be applied towards early detection, screening and disease progression of OPML and malignant lesion, these assay systems can be used for patients susceptible to or diagnosed with oral cancer.
[0170] Merely for illustration, only representative number/type of graph, chart, block, and sub-block diagrams were shown. Many environments often contain many more block and sub-block diagrams or systems and sub-systems, both in number and type, depending on the purpose for which the environment is designed.
[0171] While specific embodiments of the invention have been shown and described in detail to illustrate the inventive principles, it will be understood that the invention may be embodied otherwise without departing from such principles.
[0172] Reference throughout this specification to “one embodiment”, “an embodiment”, or similar language means that a particular feature, structure, or characteristic described in connection with the embodiment is included in at least one embodiment of the present invention. Thus, appearances of the phrases “in one embodiment”, “in an embodiment” and similar language throughout this specification may, but do not necessarily, all refer to the same embodiment.
[0173] It should be understood that the figures and/or screen shots illustrated in the attachments highlighting the functionality and advantages of the present invention are presented for example purposes only. The present invention is sufficiently flexible and configurable, such that it may be utilized in ways other than that shown in the accompanying figures.
[0174] It should be understood that the examples and embodiments described herein are for illustrative purposes only and that various modifications or changes in light thereof will be suggested to persons skilled in the art and are to be included within the spirit and purview of this application and scope of the appended claims. All publications, patents, and patent applications cited herein are hereby incorporated by reference in their entirety for all purposes.
[000] References
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,CLAIMS:CLAIMS
I/ WE CLAIM:
1) A method for detecting or diagnosing or predicting oral potentially malignant (OPML) and malignant lesions in a subject having or at risk of developing OPML, the method comprising:
a. obtaining a sample from the subject;
b. identification of selected proteins that are differentially regulated in a sample, wherein the protein is selected from the group comprising of CD44, CYCLIN D1, SNA-1, MAA, and combinations thereof; and
c. comparing the cytology of the samples; wherein the levels of SNA-1, MAA, CYCLIN D1 and CD44 proteins in the patient sample, when compared to the SNA-1, MAA, CYCLIN D1 and CD44 proteins level in the control sample is/are indicative that the subject has or is at risk of developing OPML.
2) The method of claim 1, wherein the four markers were evaluated in cytology by using either HRP conjugated secondary antibodies (CD44, CYCLIN D1) or by FITC conjugation (SNA-1, MAA), wherein the level of SNA-1, MAA and CD44 in the sample is at a higher level compared to the uptake in normal oral epithelial cells.
3) The method as claimed in claim 1, wherein the sample in step a) is selected from a group consisting of oral mucosal cells, saliva, blood, and combinations thereof.
4) The method as claimed in claim 1, wherein step b) comprises carrying out immunocytochemistry, immunohistochemistry and combinations thereof.
5) A method for aiding assessment of a patient's likely severity or likelihood of premalignant lesions and cancer, or aiding in selection of a treatment regime for the patient, or aiding in assessment of a treatment regime, or the detection and diagnosis of oral or oral potentially malignant (OPML) or malignant lesions in a subject, the method comprising:
a. determining in a biological sample from the patient the amount of the expression level of at least one protein selected from the group consisting of CD44, CYCLIN D1, SNA-1, MAA and a combination thereof; and
b. comparing the determined expression levels of said protein in said biological sample with the level in a reference.
6) The method according to any one of the preceding claims, wherein the cancer is selected from the group consisting of oral potentially malignant (OPML) and Oral cancers.
7) A method of detecting pre-cancer/cancer in a patient, the method comprising of collecting the cells from the patient and staining the same with an anti- CyclinD1 or anti- CD44 antibodies or with SNA-1 or MAA lectins or anti- CYCLIN D1 antibody or a fragment or derivative thereof labelled with a detectable label, allowing the labelled antibody to locate to the cancer, and imaging the cancer.
8) The method according to any one of the preceding claims, wherein the detection of oral potentially malignant (OPML) and malignant lesions in cancers of oral cavity (tongue, buccal mucosa, palate, RMT, gingiva) can be detected in a minimally invasive manner, wherein the method can be extended to larynx, pharynx, oropharynx, primary or secondary cancers, apart from oral cancers in animals that includes humans as a cost-effective method for screening on large scale, for prognosis, screening at various stages of cancers in the subjects during and post -treatment, in the form of kits, slides, such other easily usable, disposable, multi-utility kits containing the preferred biomarkers for detecting preferably head and neck, oral cancers with specificity, sensitivity, accuracy, speed, reliability and reproducibility.
9) A kit for diagnosing or predicting or likelihood of progression of cancer, or aiding in selection of a cancer treatment regime for the patient, or aiding in assessment of a cancer treatment regime, wherein the kit comprising at least one reagent for determining the amount of the expression level of at least one gene selected from the group comprising of CD44, CYCLIN D1, SNA-1, MAA and a combination thereof.
10) The kit as claimed in claim 9, wherein the kit comprising of at least one reagent that specifically binds to a cancer biomarker and a package insert containing instructions using the kit, wherein the marker panel can be used for the development of a Point-of-Care assay system that can be applied towards early detection, screening, and disease progression.