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EP-4735644-A1 - BIOMARKERS AND USES THEREFOR

EP4735644A1EP 4735644 A1EP4735644 A1EP 4735644A1EP-4735644-A1

Abstract

This disclosure relates generally to biomarkers of cancer. More particularly, the present disclosure relates to miRNA biomarkers and their use in methods, compositions, apparatuses, devices and kits for determining an indicator that is useful for assessing a likelihood that an oral potentially malignant disorder or oral cancer is present or absent in a subject.

Inventors

  • PUNYADEERA, Chamindie Kusalini
  • BALAKITTNEN, Jaikrishna
  • WEERAMANGE, Sasee Chameera Ekanayake

Assignees

  • Griffith University

Dates

Publication Date
20260506
Application Date
20240628

Claims (20)

  1. 1. A method for determining an indicator used in assessing a likelihood that oral cancer or a healthy status is present or absent in a subject, the method comprising, consisting or consisting essentially of: (7) determining a biomarker value for at least one miRNA biomarker {e.g., 1, 2, 3, 4, 5, 6, 7 or 8 miRNA biomarkers) in a saliva sample obtained from the subject, wherein a respective biomarker value is indicative of a level of a corresponding miRNA biomarker in the sample, and wherein the at least one miRNA biomarker is selected from miR_7_5p, miR_10b_5p, miR_431_5p, miR_486_3p, miR_182_5p, miR_215_5p, miR_4707_3p and miR_3614_5p; and (8) determining the indicator using the biomarker value(s).
  2. 2. A method for determining an indicator used in assessing a likelihood that OPMD or OC is present or absent in a human subject, the method comprising, consisting or consisting essentially of: (9) determining a biomarker value for at least one miRNA biomarker {e.g., 1, 2, 3 or 4 miRNA biomarkers) in a saliva sample obtained from the subject, wherein a respective biomarker value is indicative of a level of a corresponding miRNA biomarker in the sample, and wherein the at least one miRNA biomarker is selected from miR-7-5p, miR- 10b-5p, miR-215-5p and miR-4707-3p; and (10) determining the indicator using the biomarker value(s).
  3. 3. A method for determining an indicator used in assessing a likelihood that OPMD or a healthy status is present or absent in a human subject, the method comprising, consisting or consisting essentially of: (11) determining a biomarker value for at least one miRNA biomarker {e.g., 1, 2, 3 or 4 miRNA biomarkers) in a saliva sample obtained from the subject, wherein a respective biomarker value is indicative of a level of a corresponding miRNA biomarker in the sample, and wherein the at least one miRNA biomarker is selected from miR-10b-5p, miR-182-5p, miR-215-5p and miR-3614-5p; and (12) determining the indicator using the biomarker value(s).
  4. 4. The method of claim 1 or claim 2, wherein the subject has at least one clinical sign of OC, which is preferably selected from: lip or mouth sore that does not heal; a white or reddish patch on the inside of the mouth; loose teeth; a growth or lump inside the mouth; mouth pain; ear pain; and difficult or painful swallowing.
  5. 5. The method of claim 2 or claim 3, wherein the subject has at least one clinical sign of OPMD, which is preferably selected from: persistent white patch in the oral cavity that cannot be rubbed off, which is generally asymptomatic; persistent white and red patch that cannot be rubbed off, typically with some discomfort; fiery red patch with discomfort, tingling and/or sensitivity to touch, hot beverages or spicy foods; corrugated white patches; keratotic striae or white plaque, which are typically asymptomatic; ulcerative striae, which are typically sore and painful; burning sensation to spicy food; and restricted mouth opening.
  6. 6. The method of any one of claims 2, 3 and 5, wherein the subject has a condition associated with increased risk of OPMD, which is preferably selected from: oral leukoplakia; erythroplakia; proliferative verrucous leukoplakia (PVL); palatal lesions in reverse smokers; lupus erythematosus; dyskeratosis congenita; epidermolysis bullosa; actinic cheilitis; chronic hyperplastic candidiasis; exophytic verrucous hyperplasia; oral lesions of graft vs host disease; oral submucous fibrosis; oral lichen planus and oral lichenoid lesion/ reaction.
  7. 7. The method of any one of claims 1 to 3, wherein the subject is asymptomatic but at risk of developing OC or OPMD, wherein the at-risk subject preferably has any one or more of: a history of smoking or oral tobacco use: a history of drinking mate (/.e., a stimulant drink common in South America); a history of chewing betel quid (/.e., a stimulant commonly used in parts of Asia); are overweight; have Fanconi anemia and/or dyskeratosis congenita.
  8. 8. The method of any one of claims 1 to 7, further comprising applying a function to biomarker values to yield at least one functionalized biomarker value and determining the indicator using the at least one functionalized biomarker value.
  9. 9. The method of claim 8, wherein the function includes normalization of biomarker values, and the level of a miRNA biomarker is normalized against a housekeeping biomarker (e.g., an RNA housekeeping biomarker, representative examples of which are selected from miR-191-5p, miR-484 and SNORD96A).
  10. 10. An apparatus for determining an indicator used in assessing a likelihood that OC or a healthy status is present or absent in a human subject, the apparatus comprising at least one electronic processing device that: • determines a biomarker value for at least one miRNA biomarker (e.g., 1, 2, 3, 4, 5, 6, 7 or 8 miRNA biomarkers) in a saliva sample obtained from the subject, wherein a respective biomarker value is indicative of a level of a corresponding miRNA biomarker in the sample, wherein the at least one miRNA biomarker is selected from miR_7_5p, miR_10b_5p, miR_431_5p, miR_486_3p, miR_182_5p, miR_215_5p, miR_4707_3p and miR_3614_5p; and • determines the indicator using the derived biomarker value(s).
  11. 11. An apparatus for determining an indicator used in assessing a likelihood that OPMD or OC is present or absent in a human subject, the apparatus comprising at least one electronic processing device that: • determines a biomarker value for at least one miRNA biomarker (e.g., 1, 2, 3 or 4 miRNA biomarkers) in a saliva sample obtained from the subject, wherein a respective biomarker value is indicative of a level of a corresponding miRNA biomarker in the sample, wherein the at least one miRNA biomarker is selected from miR-7-5p, miR-10b-5p, miR-215-5p and miR-4707-3p; and determines the indicator using the derived biomarker value(s).
  12. 12. An apparatus for determining an indicator used in assessing a likelihood that OPMD or a healthy status is present or absent in a human subject, the apparatus comprising at least one electronic processing device that: • determines a biomarker value for at least one miRNA biomarker {e.g., 1, 2, 3 or 4 miRNA biomarkers) in a saliva sample obtained from the subject, wherein a respective biomarker value is indicative of a level of a corresponding miRNA biomarker in the sample, wherein the at least one miRNA biomarker is selected from miR-10b-5p, miR-182-5p, miR-215-5p and miR-3614-5p; and • determines the indicator using the derived biomarker value(s).
  13. 13. A composition comprising a mixture of a DNA polymerase, salivary miRNA derived cDNA from a human subject, suitably with at least one clinical sign of OC or at risk of OC, wherein the salivary miRNA derived cDNA comprises at least one cDNA biomarker (e.g., 1, 2, 3, 4, 5, 6, 7 or 8 cDNA biomarkers) selected from miR_7_5p cDNA, miR_10b_5p cDNA, miR_431_5p cDNA, miR_486_3p cDNA, miR_182_5p cDNA, miR_215_5p cDNA, miR_4707_3p cDNA and miR_3614_5p cDNA, and wherein the composition further comprises for a respective cDNA biomarker at least one oligonucleotide primer or probe that hybridizes to the cDNA biomarker.
  14. 14. A composition comprising a mixture of a DNA polymerase, salivary miRNA derived cDNA from a human subject, suitably with at least one clinical sign of OPMD or OC or at risk of OPMD or OC, wherein the salivary miRNA derived cDNA comprises at least one cDNA biomarker (e.g., 1, 2, 3 or 4 cDNA biomarkers) selected from miR-7-5p cDNA, miR- 10b-5p cDNA, miR-215-5p cDNA and miR-4707-3p cDNA, and wherein the composition further comprises for a respective cDNA biomarker at least one oligonucleotide primer or probe that hybridizes to the cDNA biomarker.
  15. 15. A composition comprising a mixture of a DNA polymerase, salivary miRNA derived cDNA from a human subject, suitably with at least one clinical sign of OPMD or at risk of OPMD, wherein the salivary miRNA derived cDNA comprises at least one cDNA biomarker (e.g., 1, 2, 3 or 4 cDNA biomarkers) selected from miR-10b-5p cDNA, miR-182-5p cDNA, miR-215-5p cDNA and miR-3614-5p cDNA, and wherein the composition further comprises for a respective cDNA biomarker at least one oligonucleotide primer or probe that hybridizes to the cDNA biomarker.
  16. 16. A device for nucleic acid amplification of salivary miRNA derived cDNA, the device comprising a plurality of reaction vessels, individual reaction vessels comprising the composition of any one of claims 13 to 15.
  17. 17. A method for inhibiting the development or progression of OC in a subject, the method comprising: exposing the subject to a treatment regimen for OC at least in part on the basis that the subject is determined by the indicator-determining method of any of claims 1, 2, 4, 5, 10 and 11 as having a likelihood of a presence of OC.
  18. 18. A method for inhibiting development of OC or progression of OPMD to OC in a subject, the method comprising: exposing the subject to a treatment regimen for OPMD at least in part on the basis that the subject is determined by the indicator-determining method of any of claims 2, 3 and 6 to 11 as having a likelihood of a presence of OC.
  19. 19. A method for managing treatment of a subject, the method comprising: not exposing the subject to a treatment regimen for OC or OPMD at least in part on the basis that the subject is determined by the indicator-determining method of any one of claims 1, 3 and 4 to 11 as having a likelihood of a presence of a healthy status.
  20. 20. A kit for determining an indicator used in assessing a likelihood that OC or a healthy status is present or absent in a subject, the kit comprising: for each of at least one miRNA biomarker (e.g., 1, 2, 3, 4, 5, 6, 7 or 8 miRNA biomarkers) at least one oligonucleotide primer and/or at least one oligonucleotide probe that hybridizes to the miRNA biomarker or to a cDNA corresponding to the miRNA biomarker, wherein the at least one miRNA biomarker is selected from miR_7_5p, miR_10b_5p, miR_431_5p, miR_486_3p, miR_182_5p, miR_215_5p, miR_4707_3p and miR_3614_5p.

Description

TITLE BIOMARKERS AND USES THEREFOR' RELATED APPLICATIONS [0001] This application claims priority to Australian Provisional Application No. 2023902061 entitled "Biomarkers and uses therefor" filed 29 June 2023, the contents of which are incorporated herein by reference in their entirety. FIELD [0002] This disclosure relates generally to biomarkers of cancer. More particularly, the present disclosure relates to miRNA biomarkers and their use in methods, compositions, apparatuses, devices and kits for determining an indicator that is useful for assessing a likelihood that an oral potentially malignant disorder or oral cancer is present or absent in a subject. BACKGROUND [0003] Oral cancer (OC) is the sixteenth most common cancer worldwide, with a high prevalence in particular regions and certain ethnicities with predisposing lifestyles. In particular, two thirds of the cases are from developing countries, with the highest incidence rate in Papua New Guinea followed by Bangladesh and Romania (International, 2022, Available: https://www.wcrf.org/cancer-trends/mouth-and-oral-cancer-statistics/). This geographical clustering is seemingly linked with high levels of tobacco and alcohol usage, which are attributed as major risk factors for more than 90% of OC cases. Oral squamous cell carcinoma (OSCC) accounts for more than 90% of all OC cases. The five-year survival rate of newly diagnosed OC patients is approximately 50% (Thavarool et al., 2019, World Journal of Surgical Oncology 17). Diagnostic delays and difficulties in seeking health assistance significantly contribute to the poor outcomes of these patients. Therefore, relevant, timely intervention may reduce the chance of loco- regional/distant metastasis and related complications (Baykul et al., 2010, J Int Med Res. 38(3):737-49). However, the early stages of OC are often asymptomatic, and current diagnostic strategies often fail to detect early malignant lesions (Baykul et al., 2010, supra). Around 8% of OC cases occur due to the malignant transformation from oral potentially malignant disorders (OPMD), a pre-cancer stage of OC (Gurizzan et al., 2021, BMC Cancer 21:561). OPMDs can present as either localized lesions or widespread lesions affecting significant portions of the oral mucosa. Studies have reported an improvement in patient survival upon early diagnosis. [0004] Currently, histopathological assessment of biopsy samples is considered the gold standard for diagnosis of OC and OPMD. However, this is painful for patients and requires the expertise to biopsy the most pathologically advanced areas in a lesion, and the expertise of anatomical pathologists to ensure the most accurate diagnosis (Su et al., 2021, Diagnostics (Basel) 11). An incisional biopsy is only representative of the exact area of the lesion being biopsied, and this limitation has encouraged scientists and clinicians to attempt alternative methods to achieve a better representative sample from a biopsy of an OPMD (Su et al., 2021, supra). In addition, less sensitive and specific methods such as vital staining, oral cytology, and optical imaging are in clinical practice, but each has limitations of its own (Walsh et al., 2021, Cochrane Database Syst Rev. 7(7):CD010276). Despite the severe health impacts of OC, there are no approved stand-alone biomarkers for the diagnosis of OC and prediction of the risk of OC development in OPMD patients. Therefore, the clinical world desperately needs a reliable and ideally non-invasive biomarker for the early diagnosis of OC and for the prediction of malignant transformation in OPMD patients that would play a decisive role in improving patient outcomes. SUMMARY [0005] The present disclosure arises from the determination that certain miRNA biomarkers from saliva have strong discrimination performance for differentiating between subjects with OC and controls, whilst others have strong discrimination performance for distinguishing between OPMD subjects and subjects with OC, and still others can be used to differentiate subjects with OPMD from controls. Based on these determinations, methods, apparatuses, compositions, devices and kits are disclosed, which take advantage of biomarkers disclosed herein to determine a likelihood that OC, OPMD, or a healthy status is present or absent in a subject. [0006] Accordingly, in one aspect, disclosed herein are methods for determining an indicator used in assessing a likelihood that OC or a healthy status is present or absent in a subject. These methods general comprise, consist or consist essentially of: (1) determining a biomarker value for at least one miRNA biomarker (e.g., 1, 2, 3, 4, 5, 6, 7 or 8 miRNA biomarkers) in a saliva sample obtained from the subject, wherein a respective biomarker value is indicative of a level of a corresponding miRNA biomarker in the sample, and wherein the at least one miRNA biomarker is selected from miR_7_5p, miR_10b_5p, miR_431_5p, miR_486_3p, miR_182_5p, miR_215_5p, miR_4707_3p