IN VITRO SCREENING METHOD AND KIT FOR EARLY DIAGNOSIS OF ORAL CAVITY TUMOURS

Abstract

The present invention falls within the field of the early diagnosis of tumours of the oral cavity. In particular, the invention relates to a method for the diagnosis and/or for predicting the risk of developing tumours of the oral cavity comprising the detection in cell extracts of certain markers of tumours of the oral cavity using immunological assays, for example ELISA (enzyme-linked immunosorbent assay). The invention also relates to the relative kit for the diagnosis and/or for predicting the risk of developing tumours of the oral cavity.

Claims

1. An in vitro method for predicting the risk of developing and/or for the diagnosis and/or for the prognosis and/or for monitoring the progression and/or for the screening of a therapeutic treatment of a tumour of the oral cavity in a subject comprising the following steps: a) detecting and/or quantifying at least the androgen receptor (AR) and the estrogen receptor (ER) in a biological sample obtained from the subject, optionally from the oral cavity of the subject; and b) comparing with respect to a control sample.

2. The method according to claim 1, wherein in step a) the epithelial growth factor receptor (EGF-R) is further detected and/or quantified.

3. The method according to claim 1, wherein the presence of AR and/or ER, and optionally EGF-R, and/or a higher amount of AR and/or ER, and optionally EGF-R, with respect to the amount in the control sample indicates that the subject is at risk of developing or is suffering from a tumour of the oral cavity.

4. The method according to claim 1, wherein step a) comprises: contacting and incubating said biological sample with at least an anti-AR antibody and an anti-ER antibody and optionally an anti-EGF-R antibody under conditions such that the AR and the ER and optionally the EGF-R bind to said antibodies and form an antibody-antigen complex if AR and/or ER and/or EGF-R are present; and detecting and/or quantifying AR and/or ER and/or EGF-R bound to the antibody, preferably using detection and/or quantification means for said antibodies.

5. The method according to claim 1, wherein the subject in whom the presence of AR and/or ER and optionally EGF-R and/or of an anti-AR antibody and/or an anti-ER antibody and optionally an anti-EGF-R antibody has been detected and/or quantified is subsequently subjected to further methods of diagnosis of a tumour of the oral cavity.

6. The method according to claim 1, wherein said biological sample originates from an inflamed area of the oral cavity.

7. The method according to claim 1, wherein said biological sample is a cellular, optionally subjected to cellular lysis, or a tissue sample or a fluid, said fluid optionally being one of saliva, blood or serum.

8. The method according to claim 1, wherein said tumour of the oral cavity is selected from the group consisting of: tumour of the oral mucosa, lingual tumour, pharyngeal tumour, laryngeal tumour, palatal tumour, tumour of the glandular epithelium, squamous cell carcinoma (SSC), epidermal carcinoma of the mouth, laryngeal carcinoma, carcinoma of the tongue and carcinoma of the lip.

9. A kit comprising: detection and/or quantification means of AR and ER and optionally EGF-R; optionally control means.

10. The kit according to claim 9 comprising: (a) at least an anti-AR antibody and an anti-ER antibody; and (b) detection and/or quantification means of at least an AR-antibody complex and an ER-antibody complex.

11. The kit according to claim 10 further comprising an anti-EGF-R antibody and detection and/or quantification means of an EGF-R-antibody complex.

12. The kit according to claim 10, wherein the anti-AR antibody, the anti-ER antibody and optionally the anti-EGF-R antibody are immobilized to a solid support.

13. The kit according to claim 9, comprising a device with two ends, wherein the first end comprises the anti-AR antibody, the anti-ER antibody and optionally the anti-EGF-R antibody and the second end comprises a brush for drawing a biological sample from a subject, optionally, the first end further comprises a positive control and/or a negative control.

14. The kit according to claim 9 comprising at least a buffer solution and/or a lysis solution and/or a detection system.

15. The kit according to claim 14 wherein said buffer solution and/or lysis solution and/or detection system are provided in different wells, optionally, placed in a single box.

16. (canceled)

17. (canceled)

18. (canceled)

19. A device comprising two ends, wherein the first end comprises the anti-AR antibody, the anti-ER antibody and optionally the anti-EGF-R antibody and the second end comprises a brush for drawing a biological sample from a subject, wherein the first end optionally further comprises a positive control and/or a negative control.

20. (canceled)

21. An in vitro method for predicting the risk of developing and/or for the diagnosis and/or for the prognosis and/or for monitoring the progression and/or for the screening of a therapeutic treatment of a tumour of the oral cavity in a subject comprising: a) immersing a biological sample in a lysis solution to obtain a first solution in which the androgen receptor (AR) and/or the estrogen receptor (ER) and/or epithelial growth factor receptor (EGF-R) are released, if present in the biological sample; b) immersing the anti-AR and anti-ER antibodies and optionally anti-EGF-R in said first solution to obtain first antibody-antigen complexes; c) immersing said first antibody-antigen complexes in a second solution comprising a secondary antibody provided with an enzymatic detector system to obtain second antibody-antigen complexes; d) immersing said second antibody-antigen complexes in a third solution comprising a substrate for the detection of the secondary antibody; and e) detecting and/or quantifying the AR and/or ER and/or EGF-R with respect to a control sample.

Description

[0066] The present invention will be described through non-limiting examples, making reference to the following figures:

[0067] FIG. 1. Components: device provided with a brush (side a) and PVDF strip on plastic support (side b) and detection box.

[0068] FIG. 2. Components: device provided with a brush (side a) and PVDF strip on plastic support armed with primary antibodies (side b) and detection box with twenty wells with buffer, lysing and detection systems useful for two patients.

[0069] FIG. 3. Operating scheme of the device: (a) drawing of the cells from the oral mucosa with the brush; (b) immersion of the brush in the first well.

[0070] FIG. 4. Scheme of use of the device: (a) extraction of the brush; (b) extraction of the PVDF strip.

[0071] FIG. 5. Scheme of immersion of the PVDF strip in the first well and subsequent four washes.

[0072] FIG. 6. Scheme of immersion of the strip in well 6 for the reaction with the secondary antibody, in wells 7, 8 and 9 for the washes and in well 10 for the detection with the substrate.

[0073] FIG. 7. Colorimetric expression relative to the results.

[0074] FIG. 8. Steps of preparing the strip.

[0075] FIG. 9. (a) and (b) Strip formation scheme and (c) subsequent development.

[0076] FIG. 10. Western blot for EGFR in KB and HEP-2 cells.

[0077] FIG. 11. Western blot for AR in KB and HEP-2 cells.

[0078] FIG. 12. Western blot for ER in KB and HEP-2 cells.

MATERIALS AND METHODS

Protocol for Preparing the PVDF Strip

[0079] The steps for preparing the PVDF strip armed with the primary antibodies of interest are the following:

[0080] 1) Hydration of the PVDF strip (ThermoFisher Scientific Catalog Number LC2002) with methanol for 5 minutes (FIG. 8, Panel A).

[0081] 2) Wash with water for 5 minutes (FIG. 8, Panel B).

[0082] 3) Two washes with PBS for 5 minutes (FIG. 8, Panel B).

[0083] 4) Incubation with the protein A-biotin from Staphylococcus aureus (Sigma-Aldrich, 10 μg/mL) for 1 hour in PBS (FIG. 8, Panel B).

[0084] 5) Two washes with PBS (FIG. 8, Panel B).

[0085] 6) Blocking with 3% BSA solution in PBS for 1 hour (FIG. 8, Panel B).

[0086] 7) Three washes with PBS for 5 minutes (FIG. 8, Panel B).

[0087] 8) Mounting of the PVDF on BioRad multi-channel apparatus (FIG. 8, Panels C, D, E).

[0088] 9) Incubation with solution of rabbit antibodies (anti-AR antibody: Ab N-20 sc 816; Santa Cruz Biotechnologies Inc., Santa Cruz, Calif.; anti-ERα antibody sc 543; Santa Cruz Biotechnologies Inc., Santa Cruz, Calif.; and anti-EGFR antibody sc-03-G, Santa Cruz Biotechnologies Inc., Santa Cruz, Calif.) about 400 μL per channel, under stirring O.N. (FIG. 8, Panel F).

[0089] 10) Three washes with PBS for 5 minutes in every channel (FIG. 8, Panel F).

[0090] 11) Two washes of the filter with PBS TEA 0.2 M (FIG. 8, Panel F).

[0091] 12) Incubation with DMP 25 mM in TEA HCl 0.2 M pH 8.2 (FIG. 8, Panel F, G, H)

[0092] 13) Incubation with TEA 0.2 M+20 mM ethanolamine (FIG. 8, Panel F, G, H)

[0093] 14) Two washes with PBS for 5 minutes (FIG. 8, Panel F, G, H).

[0094] 15) Storage in 0.02 NaN3 in PBS (FIG. 8, Panel F, G, H).

[0095] 16) The filter is cut (FIG. 9a) perpendicularly to the channels in which the primary anti-EGFR, anti-AR and anti-ER antibodies were loaded (for the capture of the relative antigens released from the collected cytology sample) to obtain a strip about 0.4 cm wide which will be mounted on the support of the device (FIG. 9b). The strip is then treated with ELISA method with the aim of revealing the specific antigens present in the tested cytology sample (FIG. 9c).

[0096] The cell lines derived from the mucosa of the oral cavity express the epithelial growth factor receptor (EGFR) and are therefore not suitable as negative control of a screening based on the positivity of the expression of hormone receptors.

[0097] The loaded negative control (CTR−) derives from clones of tumour cells deriving from mammary cancer (MDA-MB-453, ATCC HTB-131), whose specific characteristics are listed below:

[0098] i) the clone, MDA-MB-453, despite being of epithelial tumour derivation does not express the epithelial growth factor receptor (EGF-R);

[0099] ii) the clone, MDA-MB-453, despite being of mammary tumour cell derivation, does not express the androgen receptor (AR) and the estrogen receptor (ER) (Kristina Subik et al., Breast Cancer (Auckl). 2010; 4: 35-41).

[0100] The positive control (CTR+) is an anti-actin antibody (Sigma-Aldrich, A2066).

ELISA Protocol of the PVDF Strip in Cells of the Oral Cavity

[0101] KB cells (ATCC CCL-17) derived from the epidermal carcinoma of the mouth and HEP-2 cells (ATCC CCL-23) derived from laryngeal carcinoma, are lysed in a lysis buffer, the extracted proteins are separated by SDS-PAGE. At the end of the electrophoretic run, the proteins are transferred onto a PVDF filter for 2 hours at room temperature. Afterwards, the filter is washed for two hours in a PBS buffer containing Tween-20 pH 7.2 (PBST-buffer, PBS Sigma-Aldrich P5368 and 0.02% Tween-20 Sigma-Aldrich P1379) and 3% bovine serum albumin (BSA) in order to block the nonspecific sites. The filter is subsequently incubated for at least two hours with the specific anti AR, anti ER and anti EGFR primary antibodies (anti-AR antibody: Ab N-20 sc 816; Santa Cruz Biotechnologies Inc., Santa Cruz, Calif.; anti-ERα antibody sc 543; Santa Cruz Biotechnologies Inc., Santa Cruz, Calif.; and anti-EGFR antibody sc-03-G, Santa Cruz Biotechnologies Inc., Santa Cruz, Calif.). Finally, after three 10-minute washes with PBST-buffer, the filter is incubated with the specific secondary antibodies and the signal will be detected by means of a chemiluminescence release kit (ECL, Amersham Pharmacia Inc.). The semiquantitative densitometric analysis was run by means of a Scan LKB (Amersham Pharmacia Inc.).

DESCRIPTION OF THE DEVICE

Analysis Instrumentation and Composition Thereof

[0102] 1) plastic pen-like device with two functional ends one provided with brush and yellow cap (side A) the other provided with support for the PVDF strip (Immobilon) armed with the primary antibodies of interest, with transparent cap (side B) FIG. 1 and FIG. 2;

[0103] 2) box organized in rows of 10 wells closed off with LED aluminium strips containing buffers and lysing and detection systems; FIG. 1 and FIG. 2.

Mode of Use

[0104] The dentist, in the presence of potentially cancerous lesions, can draw a sample of cells using the specific collection instrument present in the kit and proceed as follows:

[0105] 1) Collection of the cytology sample: drawing of cells from the oral cavity on the suspect mucosa with the special brush present at the end (side A) of the device provided with a yellow cap; FIG. 3a.

[0106] 2) Immersion of the brush for 15 minutes in the first well containing the lysis buffer, which enables the release in solution of the proteins, i.e. the antigens of interest, EGFR, AR and ER present in the extract of the cells of the oral cavity; FIG. 3b.

[0107] 3) The cap of the device side A is closed again; FIG. 4.

[0108] 4) The cap of the device side B is opened; FIG. 4b.

[0109] 5) Immersion of the end (side B) of the support with the PVDF strip for 5 minutes in the first well to enable the interaction of the proteins (antigens) with the primary antibodies adhered to the PVDF strip in order to form the immunocomplex (anti-AR antibody: Ab N-20 sc 816; Santa Cruz Biotechnologies Inc., Santa Cruz, Calif.; anti-ERα antibody sc 543; Santa Cruz Biotechnologies Inc., Santa Cruz, Calif.; and anti-EGFR antibody sc-03-G, Santa Cruz Biotechnologies Inc., Santa Cruz, Calif.); FIG. 5.

[0110] 6) Washes (three) of the support with the PVDF strip in wells containing PBST-buffer to eliminate the proteins non-specifically adhered to the immunocomplex; FIG. 5.

[0111] 7) Immersion of the support with the PVDF strip for 10 minutes in the well containing the secondary antibody provided with an enzymatic detector system (Alkaline phosphatase, Sigma-Aldrich A2306); FIG. 6.

[0112] 8) Washes (two) of the support with the PVDF strip in the wells containing PBST-buffer to eliminate the excess secondary antibody; FIG. 6.

[0113] 9) Immersion of the support with the PVDF strip in the well containing the substrate (BCIP/NBT, ROCHE 11681451001) necessary for the colorimetric reaction; FIG. 6.

[0114] 10) Washes (two) of the strip in the well containing PBST-buffer to eliminate the excess; FIG. 6.

[0115] 11) The colorimetric reaction will be noted in the presence of the proteins of interest; FIG. 7.

RESULTS

[0116] The proposed invention is the fruit of scientific studies carried out by the author on the crosstalk between EGF and estradiol (ER) and androgen (AR) receptors in epithelial tumours. Such studies were the translational prerequisite for the formulation of an instrument useful for the early diagnosis of neoplastic pathologies. Such use finds soundness in the results, which demonstrate that the complex of steroid receptors (AR/ER/Src) is required for the action of the EGF (1.2). The use of the ELISA techniques reported in the scientific papers of the author made the realization of the detector system of the device possible.

[0117] The cell extracts used in the laboratory selected for the validation of the conceived instrument, come from KB cells derived from epidermal carcinoma of the mouth and from HEP-2, cells derived from laryngeal carcinoma. FIG. 9c shows the results regarding the co-expression of the proteins of interest (EGFR, AR and ER), on the PVDF strip of the device. As confirmation of the results obtained with the device, the cell extracts were subjected to Western blot analysis for the individual receptors and the results are shown in FIGS. 10, 11, 12. In FIG. 10, the bands relative to the EGFR receptor in KB and HEP-2 cells are evident; the samples were loaded in triplicate and the receptor is clearly evident in the two cell types, whereas it does not appear evident in the negative control represented by MDA-MB-453 cells (Kristina Subik et al. Breast Cancer (Auckl). 2010; 4: 35-41). In FIG. 11 one sees the presence in the same cells of the AR receptor (the samples were loaded in duplicate), in this case as well the band is visible only in the cells of the oral cavity (KB and HEP-2) and not in the negative control (MDA-MB-453). Finally, FIG. 12 shows the ER receptor (the samples were loaded in duplicate) in this case as well the receptor was revealed only in the cells of the oral cavity (KB and HEP-2) and not in the negative control (MDA-MB-453).

[0118] The test proposed ensures a significantly high index of predictability, since the simultaneous expression of these three receptors correlates with the change in the cell phenotype from benign to malignant.

REFERENCES

[0119] 1) Fiorelli A, Ricciardi C, Pannone G, Santoro A, Bufo P, Santini M, Serpico R, Rullo R, Pierantoni G M, Di Domenico M. Interplay between steroid receptors and neoplastic progression in sarcoma tumors. J Cell Physiol. 2011 November; 226(11):2997-3003. doi: 10.1002/jcp.22645.

[0120] 2) Migliaccio A, Castoria G, Di Domenico M, Ciociola A, Lombardi M, De Falco A, Nanayakkara M, Bottero D, De Stasio R, Varricchio L, Auricchio F. Crosstalk between EGFR and extranuclear steroid receptors. Ann N Y Acad Sci. 2006 November; 1089:194-200.