Diagnostic methods and kits for oral cancer

Abstract

A method and kit for estimating the presence of oral squamous cell carcinoma in a subject by analyzing the protein levels of EGFR, p53, B7H6, and PdL1 in a subject's sample.

Description

Technical Field

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[0001] The present invention relates to a method carried out in vitro for determining the presence of oral squamous cell carcinoma, preferably at an early stage, based on measuring the levels of a specific series of protein markers.

Background Art

[0002] More than 90% of oral cancers are oral squamous cell carcinomas (OSCC). Oral squamous cell carcinoma (OSCC) is the sixth most common cancer in the world. The average 5-year survival rate for oral cancer is about 60% (1). Treatment of advanced oral cancer requires interventions involving resection, and the quality of life significantly deteriorates as the disease progresses. In order to reduce mortality and morbidity, it is necessary to detect oral cancer very early in order to enhance the effectiveness of available treatment methods.

[0003] To date, the most effective method for obtaining diagnostically useful tissue is biopsy. Biopsy requires surgery and is performed on lesions that are already visible in clinical examinations, so early diagnosis is impossible. To date, regardless of the presence of magnifying glasses or fluorescence systems, there is no scientifically approved system other than conventional oral clinical examinations that can detect lesions at the early stages of tumors, and the gold standard for diagnosis remains biopsy and immunohistochemical staining.

[0004] This method has several limitations. It is semi-quantitative and cannot reliably determine the absolute amount of the target. Furthermore, since the tissue is highly processed, information regarding the natural state may be lost.​​​​​​​Recent studies have suggested that molecules can be detected using cytobrushes from patients with this type of tumor, but no single biomolecule has been identified that meets the real-world requirement of accurately identifying the early onset of the disease. This highlights the need for the development of non-invasive, rapid, and effective diagnostic methods to classify and identify suspicious oral lesions. [Overview of the project]

[0007] The present invention will be described below with reference to the drawings, in non-limiting examples. [Brief explanation of the drawing]

[0008] [Figure 1] The columns show the names of various markers (first triplet: EGFR, p53, Ki67, control; second triplet: B7H6, PDL1, HLAE, control), and the rows show the acquisition times of various chemiluminescences (t1, t2, t3). The numbers represent the detected chemiluminescence values. Values ​​with an FM of 1.3 to 1.49 compared to the control group are marked in yellow, and values ​​with an FM greater than 1.49 compared to the control group are marked in red. [Figure 2] The calculated FM values ​​for each marker at three different points in time are displayed only if the FM is greater than 1. For each marker with an FM greater than 1, the sum of the FM for the three strokes is reported at the bottom of the corresponding column. [Figure 3] This table shows the total FM (Frequency Meter) of each marker in the example patient for each topological region under consideration (from top to bottom: tumor center (A), healthy tissue (B), and peripheral tissue (C)). Below that, the total FM for all markers in topological regions (A, B, C) is shown. Finally, the totals for EGFR, PDL1, and B7H6 markers only are shown. Table C shows the FM signal intensity delta, i.e., the difference between the total FM of the tumor tissue and the peripheral tissue (3,2). The corresponding graph is a pie chart showing the expression rate of each marker relative to the total signal intensity represented by the total FM of all markers in the target region. In the healthy tissue region, the FM value for all markers is 0. [Figure 4]The columns show various marker names (first triplet: EGFR, p53, Ki67, control; second triplet: B7H6, PDL1, HLAE, control), and the rows show various chemiluminescence acquisition times (t1, t2, t3). The numbers represent the measured chemiluminescence values. Values ​​with an FM of 1.3 to 1.49 compared to the control are shown in light gray, and values ​​with an FM greater than 1.49 compared to the control are shown in dark gray. [Figure 5] The calculated FM value for each marker is displayed only if it is greater than 1 at three different time points. For markers with an FM greater than 1, the sum of the FMs at the three time points is reported at the bottom of the corresponding column. [Figure 6] Biopsy sample. The table shows the total FM of each marker in the example patient for each topological region, from top to bottom: tumor center (A), healthy tissue (B), and peripheral tissue (C). Below that, the total FM of all markers is shown, and further below, the total for EGFR, PDL1, and B7H6 markers only. Finally, the FM signal intensity delta, i.e., the difference between the total FM of tumor tissue and peripheral tissue (0) and the total FM of healthy tissue (0), is shown. The graph is a pie chart showing the expression rate of each marker relative to the signal intensity shown by the total FM of all regions in the target region. In the healthy tissue region, only the value for Ki67 is shown, and Ki67 shows 100% signal. [Figure 7] This is an exploded perspective view of a longitudinal section of a disposable kit according to the present invention, showing four lines. [Figure 8] Enlarged perspective view of Figure 7. [Figure 9] A cross-sectional perspective view of the components of the kit of the present invention assembled into a configuration for use. [Modes for carrying out the invention]

[0009] [Detailed explanation] To overcome the limitations of prior art, the inventors searched, identified, and validated protein markers that can be used for non-invasive, rapid, and effective screening tests to classify and identify oral squamous cell carcinoma (OSCC), and to identify suspected oral lesions with morphologically altered tissue as precancerous conditions. Examples of such oral lesions include, but are not limited to, leukoplakia, erythroplakia, palatal smoking lesions, oral lichen planus, oral submucosal fibrosis (SMF), and discoid lupus erythematosus.

[0010] Therefore, the object of the present invention was to select appropriate markers to be detected in samples collected by a non-invasive procedure using rapid ELISA (enzyme-linked immunosorbent assay) technology, preferably which can be performed by an operator after sample collection, to confirm whether the detection of these biomarkers can distinguish OSCC lesions or precancerous lesions from surrounding healthy tissue, and to create a kit that can be used in an in vitro diagnostic method for this pathology.

[0011] In a preferred embodiment, the technique is an ELISA technique comprising colorimetric signal detection or signal detection based on emission.

[0012] As an example without limitation, both a portion of a biopsy obtained from a patient and samples obtained by non-invasive procedures are considered optimal samples, namely, samples obtained from oral smears using sterile swabs for cytological sampling (e.g., cytobrushes), saliva samples, and samples generally containing epithelial cells from the oral cavity.

[0013] The inventors selected six protein-type biomarkers from a group of different markers. - Markers already used in immunohistochemical methods for the diagnosis of oral squamous cell carcinoma (OSCC) - Markers known to be present or overexpressed in transformed cells - Markers identified as candidate molecular targets in immune checkpoint inhibitor therapy

[0014] The inventors first evaluated the expression levels of several selected markers, namely, EGFR (epidermal growth factor receptor) sequences preferably corresponding to the sequence available in the NCBI database under accession number NM-005228.3, AR (androgen receptor) sequences preferably corresponding to the sequence available in the NCBI database under accession number NM-000044.4, ER (estrogen receptor) sequences preferably corresponding to the sequence available in the NCBI database under accession number NP-001428.1, PDL1 (programmed cell death ligand 1) sequences preferably corresponding to the sequence available in the NCBI database under accession number Q9NZQ7.1, B7H6 sequences preferably corresponding to the sequence available in the NCBI database under accession number Q68D85, and Ki67 sequences preferably corresponding to the sequence available in the NCBI database under accession number P46013.2. The HLAE (Class 1 Histocompatibility Antigen) sequence preferably corresponds to the sequence available in the NCBI database under accession number P13747.4, the P16INK4A sequence preferably corresponds to the sequence available in the NCBI database under accession number P42771.2, and the p53 sequence preferably corresponds to the sequence available in the NCBI database under entry number P04637.4.

[0015] The inventors first evaluated the expression of these related markers in healthy patients or volunteer patients with common oral inflammations (such as leukoplakia and lichen). All markers were protein-type and were analyzed using ELISA, particularly quantitative sandwich ELISA.

[0016] Samples were collected using a cytobrush after rinsing the patient's oral cavity with saline solution, and then transported to the laboratory in a sealed refrigerated container. At the time of analysis (within 72 hours), a lysis solution was added, and each marker was evaluated by binding it to an antibody sandwich (rabbit polyclonal antibody immobilized on a PVDF membrane and a specific mouse monoclonal antibody).

[0017] Each of the dissolved samples was incubated with a PVDF membrane pre-loaded with the corresponding polyclonal antibody for recognizing the target marker. A sandwich ELISA test was performed by detecting chemiluminescence signals using a luminol reagent (ECL, Amersham Cytiva). The light intensity emitted by each specific immune reaction was detected and analyzed using dedicated software (fiji app software).

[0018] In this preliminary study, it was shown that some tumor markers were not expressed in healthy patients and were either not expressed or had low expression in patients with oral inflammation, indicating that they are specific to certain situations of cancer, as suggested in the literature. The EGFR, p53, Ki67, B7H6, PDL1, and HLA-E markers corresponded to this. On the other hand, other markers (AR, ER, p16) showed high expression in healthy patients and at least higher expression than in patients with oral infections, but generally did not show specificity for inflammatory diseases, so they were excluded from more specific studies to be conducted later.

[0019] Therefore, the following factors were selected as the subjects of detailed research. EGFR - The epidermal growth factor receptor (EGFR) is a member of the ErbB tyrosine kinase (TK) receptor family. p53 - The cellular tumor antigen p53 acts as a tumor suppressor in many types of tumors and induces growth arrest or apoptosis depending on the physiological situation and cell type. Ki67 - It is a factor necessary to keep individual mitotic chromosomes dispersed in the cytoplasm after the nuclear membrane is disassembled. PD-L1 - Programmed cell death ligand 1 is a factor that plays an important role in the induction and maintenance of self-immune tolerance. HLA-E is a class 1 non-classical MHC that is ubiquitously expressed in hematopoietic cells and is sensitive to inflammatory signals. HLA-E binds to the CD94 / NKG2A heterodimer complex. B7-H6 is a chimeric antigen expressed in some primary tumors and is recognized by the NKp30 receptor expressed on T cells (NK cells).

[0020] These markers were selected for detailed study in various stages of oral cancer (10 patients with tumors of stages T1 to T4) and were analyzed by quantitative ELISA using an automatic signal detection device. For each patient, three different samples were collected from three topological regions in the oral cavity (tumor center region, tumor margin, and healthy tissue) using a cytobrush. Samples with a light intensity threshold less than 0.2 or greater than 20 were excluded from the results. This value is a dimensionless number given by the average light intensity of the region considered by the software. Each pixel of the region has a BLUE value from 0 to 85, and the software averages all the pixels. Samples deteriorated due to heat or transport problems were excluded.

[0021] The light intensity generated by the immune reaction was represented by a dimensionless number calculated using specific software as detailed in the examples and was evaluated for each marker and control. In each test, the intensities at three different times (denoted as T1, T2, T3) were measured, and the trend of the signal over time was analyzed. If the intensity value of a marker was higher than that of the control at all three times, that marker was considered an expressed marker, and its FM (multiplication coefficient) was calculated. The value of the multiplication coefficient is given by the ratio of the intensity value of the test channel at time t to the intensity value of the control at the same time.

[0022] As a result of the internal control test, when the FM value is less than 1.2, a non-significant signal (the corresponding signal value is not color-coded in Figure 1), when the FM value is 1.3 - 1.49, a medium signal value (the corresponding signal value is shown in light gray in Figure 1), and when the FM value exceeds 1.49, a normal signal (the corresponding signal value is shown in dark gray in Figure 1) were assigned.

[0023] For each marker, the sum of FM at the three time points considered was calculated and is reported in Figure 2 below.

[0024] The sums of each channel at the three time points calculated earlier are shown in Figure 3 and were used to calculate the total signal intensity obtained by the single sum of the six markers analyzed (FM EGFR + p53 + Ki67 + B7H6 + PDL1 + HLAE). This operation was performed for each topological region under consideration (tumor center, surface panel A, healthy tissue panel B, marginal panel C), and the results are shown in percentage format in each graph.

[0025] In Figures 3A and 3C, further analysis was performed considering only the signal intensities of the triplet EGFR, B7H6, and PDL1, which are considered to be the most important markers in the differential diagnosis of oral cancer, in addition to the total signal intensity.

[0026] Finally, the difference delta (numerical ratio) between the signal intensity of the EGFR-B7H6-PDL1 triplet in the tumor center and the signal intensity of the same reference triplet in the margin is reported in Panel C, showing that it is more highly expressed in the tumor center sample.

[0027] To verify the results obtained, and because additional material was available in the form of small biopsy fragments for some oral cancer patients already analyzed by brush biopsy, the inventors also performed ELISA analysis on this different supply material. The results regarding the expression markers obtained were compared with those from the same patients and the same phase region for brush biopsy. The samples were analyzed for the same markers obtained from brush biopsy using the same sandwich ELISA method. The qualitative and quantitative analyses at this stage, as well as the parameters considered, were the same as those described in the paragraph concerning the study performed on 10 oral cancer patients analyzed by brush biopsy. The results are shown in Figures 4, 5, and 6. See above.

[0028] These studies evaluated the markers EGFR, p53, Ki67 (the first three markers in this order shown in Figure 1, and the control for the last channel) and B7H6, PDL1, HLAE (the second three markers shown in Figure 1, and the control) in tumor tissue, margins, and healthy tissue, focusing on which markers had higher intensity values ​​than the control, and if so, what the corresponding FM was.

[0029] In all samples taken from the tumor center, all six target markers showed superior chemiluminescence signals compared to the control, and a phenotype characterized by overexpression of the six markers was defined, consistent with histological examination and tumor reporting.

[0030] In all samples taken from healthy tissue, chemiluminescent signals were generally absent for all six target markers. This was defined as a phenotype in which the expression of all six markers was absent (generally a maximum of two out of six), which is consistent with the clinical findings in healthy tissue.

[0031] In peripheral tumor lesion samples, chemiluminescent signals were observed with 4 to 6 markers, and the phenotype was defined as being consistent with clinical specimens distal to the tumor center, both in terms of the quality of luminescence and the decrease in signal intensity.

[0032] The sum of FM for each marker, represented numerically in Figure 3, tended to be higher in the central region of the lesion (e.g., EGFR marker 7.9 in the example in Figure 3) compared to the peripheral region (EGFR 5.6 in the example in Figure 3) and healthy tissue (0). In particular, the sum of FM for all six markers was significantly higher in the sample taken from the center of the lesion (51.3 in the example in Figure 3) than in the peripheral region (21.7 in the example in Figure 3) and healthy tissue (0).

[0033] Considering only the three markers EGFR, B7H6, and PDL1, the sum of the FM of these three markers is more pronounced relative to the margin (9.4) and healthy tissue (0) than the sum of all six markers in tumor tissue (29.9 in the example in Figure 3), and shows a clear intensity delta (3.2 relative to the margin in the example in Figure 3).

[0034] Studies conducted on cancer samples analyzed by biopsy showed results comparable to those conducted on the same studies on cytobrush samples. Specifically, the analysis showed a pathological sample phenotype expressing all six markers, a borderline sample phenotype represented by the expression of four, five, or in very rare cases, all six markers, and a healthy tissue phenotype where all six markers were absent or up to two markers were expressed. An example in the figure shows a healthy sample expressing only the Ki67 marker, a borderline sample overexpressing four markers, and tumor tissue overexpressing all six markers. The total FM intensity of the tumor tissue is 30, 15.5 in the borderline, and 4.3 in the healthy tissue. Therefore, the difference delta is zero both between tumor and healthy tissue, and between tumor and borderline tissue. In general, the results obtained between healthy, borderline, and pathological tissues from cancer samples taken by cytobrush showed a high degree of correspondence for biopsy fragments analyzed in the same topological region. After analyzing research results related to six markers and considering the insights gained from the scientific literature, it was decided to exclude two more markers. The excluded markers were Ki67 and HLAE. In the study patients, Ki67 is present in very high concentrations not only in the lesion center but also in healthy tissue, making differential analysis between different samples impossible if only the problematic marker is considered. The literature suggests that HLA-E is more suitable as a marker indicating the effectiveness of cancer treatment than as a diagnostic marker. Like PDL1, HLA-E is a ligand that suppresses the immune response and is usually low-expressed in tissue cells. It binds to the CD94 / NKG2A receptor complex present on NK cells of the immune system, recognizing NK cells as "self" and not attacking them. Some tumors overexpress HLA-E as a defense system against NK cells to prevent them from being recognized as foreign. In recent years, cancer treatment based on the role of HLA-E has been advancing. In fact, the use of specific monoclonal antibodies is becoming increasingly common. This inhibits the binding of HLA-E expressed by tumor cells to HLA-E receptors on NK cells, thereby destroying tumor cells.One example of a monoclonal antibody used is monalizumab, whose effectiveness has been validated by numerous studies and trials. However, not all tumors employ this mechanism, and some dysplasias lack the HLA-E ligand, necessitating a re-evaluation of this ligand as a diagnostic marker for the disease state. Based on these premises, four markers—EGFR, P53, B7H6, and PDL1—were selected as diagnostic markers for oral squamous cell carcinoma (OSCC). These markers have demonstrated an effective diagnostic role, are supported by scientific literature, and are already used in common clinical tests for tumor diagnosis.

[0035] These data suggest that this rapid and non-invasive phenotypic analysis technique can support clinical practice and provide accurate indicators of oral lesion characteristics.

[0036] Accordingly, the present invention relates to an in vitro method for the diagnosis and / or prognosis of oral tumors, preferably oral squamous cell carcinoma, preferably in an early stage. The method of the present invention is based on the selection of a protein marker suitable for the purpose, and the measurement of protein marker levels in a sample isolated from an oral tissue biopsy or oral cell sample, for example using a cytobrush, and a relative comparison of said levels with the same marker levels in a non-tumor sample isolated from a healthy individual. Furthermore, the present invention relates to a kit for carrying out the above method.

[0037] Therefore, the object of the present invention is a method for in vitro diagnosis and prognosis determination of oral squamous cell carcinoma, comprising the following basic steps. - Prepare a sample isolated from the subject (individual). - Analyze the protein levels of EGFR, p53, B7H6, and PdL1. - Compare the aforementioned levels with the levels of the corresponding proteins in samples isolated from subjects in the control group and / or the levels recorded in the reference database. Here, the increase in the aforementioned protein levels in the sample indicates the presence of oral squamous cell carcinoma in the subject.

[0038] In a preferred embodiment, this method is carried out using a qualitative ELISA method, preferably on the patient side, and preferably with dried or lyophilized reagents. These reagents are returned to solution and / or mixed at the time of use to detect and / or quantify EGFR, p53, B7H6, and PdL1 in the biological sample obtained from the subject by obtaining immediate qualitative chemimetric measurements, bioluminescence reactions, or digital detection.

[0039] In a preferred embodiment, this method is carried out using a qualitative ELISA method, and the process also includes a specific automated processing device capable of detecting and / or quantifying colorimetric, luminescence, or bioluminescent type signals.

[0040] In a preferred embodiment, the method is carried out using a quantitative ELISA method, preferably with dried or lyophilized reagents that are dissolved and / or mixed at the time of use, using a specific automated processing apparatus capable of detecting and / or quantifying colorimetric, luminescent, or bioluminescent signals, to detect and / or quantify EGFR, p53, B7H6, and PdL1 in a biological sample obtained from a subject, to obtain an immediate qualitative chemical colorimetric, bioluminescent, or digital response.

[0041] In a preferred embodiment, the present invention relates to a method for diagnosing and / or predicting the risk of developing oral squamous cell carcinoma in a subject, which can also be performed in an out-of-laboratory environment, i.e., "patient-side," and includes in vitro detection of markers EGFR, p53, B7H6, and PdL1 in a sample isolated from the subject using an ELISA assay.

[0042] The present invention also relates to related kits for the diagnosis and / or prediction of the risk of developing oral squamous cell carcinoma. The kits according to the present invention use reagents that are stable at room temperature and are rapid, highly sensitive, specific, portable, low-cost, and non-invasive. In particular, the kits of the present invention clearly ensure signal specificity and sensitivity by using a dual primary antibody, enabling the measurement of EGFR, p53, B7H6, and PdL1 markers. Specifically, a first series of rabbit polyclonal primary antibodies against the markers, attached to a PVDF membrane, and a second series of mouse monoclonal primary antibodies against the markers are used in vitro. Finally, an enzyme signal amplification system, in particular an anti-mouse secondary antibody conjugated to alkaline phosphatase or peroxidase, is used as the third series.

[0043] Accordingly, an object of the present invention is a disposable kit for testing for the presence of four antigens, comprising a base having at least a series of first and second recesses perforated downward, and a cover having a series of third and fourth recesses perforated upward. The recesses are positioned such that, when the base is covered with the cover, the first and second sets of recesses accommodate the third and fourth sets of recesses, the first or third set of recesses containing a first selective biological molecule for selecting the antigen, preferably an antibody, and a catalytic molecule bound to the selective biological molecule, such as an HRP enzyme, and the second and fourth sets of recesses containing precursors for the corresponding first and second sets of chemical reactions catalyzed by the catalytic molecule to produce a bioluminescent effect, such as hydrogen peroxide and luminol. The kit further comprises a membrane on which a series of second selective biological molecules for selecting and immobilizing the antigen onto the membrane are arranged.

[0044] In one embodiment, the kit includes at least four different analytical lines corresponding to each of the four markers, these lines having adjacent, preferably aligned, recessed arrangements. In a preferred embodiment, positive and / or negative control lines are also included.

[0045] Figure 7 shows a kit including four lines (control lines are not shown because the perspective view is a longitudinal section) for ease of interpretation. The kit comprises a base 2 having a plurality of first and second recesses 3, 3' (each recess corresponding to a stage of checking for the presence of a marker) and a cover 4 having a plurality of third and fourth recesses 5, 5', the base 2 and cover 4 are provided with connecting parts P1, P2 of corresponding shapes, and the cover is attached to the base 2 in a unique manner. The first and second recesses 3, 3' are also positioned to fit into the corresponding third and fourth recesses 5, 5'. Thus, the third and fourth recesses are also associated with the stage of checking for the presence of a marker, similar to the plurality of first and second recesses 3, 3'.

[0046] Preferably, the base 2 and / or cover 4 are constructed to hold all or part of the substance required for the test inside through one or more peelable or hand-pierced layers designed to close and / or seal one or more recesses. Before the test is performed, these layers are completely or partially removed, for example, by hand or with a piercer, so that the liquid inside the cover 4 falls into the recess of the base 2 by gravity. The peelable layers are formed of, for example, a polymer material, a metallic material, or a combination thereof.

[0047] Preferably, in the illustrated embodiment, the molded joints P1 and P2 are, for example, recesses defined on the base 2 and corresponding protrusions defined on the cover 4, but the reverse is also possible.

[0048] Furthermore, in order to reduce manufacturing costs while maintaining a high degree of chemical inertness between the reactants and the recesses 3, 3', 5, and 5' that contain them, the base 2 and cover 4 are preferably made of a plastic material such as a thermoformed polystyrene film to obtain the desired shape. As shown in the drawing, the base 2 and cover 4 made of thermoformed film define the test tray as a whole. The plastic material of the cover 4 is preferably hydrophobic, so that the liquid reagent falls completely into the recess of the base 2 that contains the corresponding powder reagent.

[0049] According to the present invention, at least one pair of first recesses 3 and third recesses 5 together contain a selective biological molecule, such as an antibody, that selectively interacts with a target antigen and even binds to multiple parts of the same antigen, and a molecule that catalyzes a chemical reaction to produce a bioluminescent substance bound to the selective biological molecule, such as the enzyme HRP peroxidase. Furthermore, another pair of second recesses 3' and fourth recesses 5' contain precursors necessary for the generation of a chemical reaction having a bioluminescent effect, which is catalyzed by the catalytic molecule. For example, precursors of substances that produce a bioluminescent effect are hydrogen peroxide and 5-amino-2,3-dihydro-1,4-phthalazinedione, i.e., luminol. Preferably, in order to enhance the efficiency of the bioluminescent effect after storage at room temperature for a certain period of time, at least one of the precursors, such as an organic precursor such as luminol, is biphasic and stored in a biphasic state within the kit. Each stage is joined by breaking or drilling holes in special recesses of the base 2 or cover 4.

[0050] Thus, the contents of the first and third recesses 3 and 5, and the contents of the second and fourth recesses 3' and 5', are configured to selectively bind to a target antigen and produce a substance that exhibits a bioluminescent effect bound to the selected antigen by causing a chemical reaction. In particular, the catalyst molecule is bound to a selective biomolecule, and both exhibit the same localization, for example, in a liquid. The substances in base 2 and cover 4 interact with the functionalized membrane during testing, and the substances fall onto the membrane by gravity, as will be described later. Furthermore, the contents of the first and third recesses 3 and 5 are biphasic in order to preserve the molecules for a long period of time and maintain the binding that enables the selective action of the antigen and the localization of the substance that produces the bioluminescent effect for as long as possible. For example, the selective biomolecule and the precursor molecule bound to it are dried to prepare a liquid suspension mixture, preferably consisting of an emulsifier and a saline buffer, for example, an emulsifier based on polysorbate and phosphate-buffered saline. Furthermore, the second and fourth recesses 3' and 5' identify two compartments for accommodating the corresponding precursors.

[0051] Therefore, the bottoms of the third and fourth recesses 5, 5' can be weakened by hand using a pointed tool (not shown) made of, for example, plastic material, or holes can be made in them. In this way, the substance contained in the corresponding first and second recesses 3, 3' can be dropped downward by gravity, particularly in a pre-measured amount, to obtain the desired mixture.

[0052] Furthermore, after the first and second recesses 3 and 3' are filled, functionalized membranes pre-exposed to a biological substance containing an antigen are sequentially placed beneath each recess to facilitate antigen selection and fixation by antibodies.

[0053] Preferably, the bioluminescent effect obtained on the membrane is captured by an image sensor and then processed by an imaging algorithm. For example, the background (dark) of pixels associated with a background label and the pattern (bright) of pixels associated with an activation label obtained by the bioluminescent effect are separated by binarization, and the activated pixels of the pattern are counted to obtain a quantitative indicator of antibody-bound antigen.

[0054] Furthermore, according to a preferred embodiment, the kit includes a reference membrane 8 containing a third selective biological molecule that is not shown for the target antigen. Meanwhile, the corresponding recess contains the substance of the recess corresponding to the test membrane. Thus, the kit includes a substance that, after the chemical reaction of the precursor, enables the production of a substance that produces a comparative bioluminescent effect, and the image of that substance enables the definition of a comparative term of the image with respect to the substance having a bioluminescent effect produced in the presence of the antigen, which is useful for performing an image filtering algorithm, as will be described in more detail below.

[0055] Furthermore, the kit may include starting material recesses 10, 11 on the corresponding base 2 and cover 4 that contain the starting material of a biological test sample containing the target antigen, for example, a biological molecule derived from an oropharyngeal swab. For example, the starting material includes a dry protease inhibitor contained in one of the starting material recesses and a dissolving solution, preferably based on physiological saline buffer. The starting material recess 10 may also be perforated to allow the two substances to be mixed after perforation, preferably with the dissolving solution positioned above the protease inhibitor.

[0056] Preferably, to wash away molecules not bound to the antigen, the base 2 comprises a washing well 12 containing a substance that can remove nonspecific binding on the membrane by substances coming from the recesses and not bound to the antigen. For example, the washing substance contains an emulsifier and a saline buffer, such as a polysorbate emulsifier and a phosphate saline buffer, where the proportion of saline buffer is greater than the proportion in the suspension. For example, the proportion of buffered saline in the washing substance is 1%, and the proportion of buffered saline in the suspension is 0.1%.

[0057] In a preferred embodiment, the disposable kit 1 is configured to perform the ELISA procedure sequentially and further comprises at least first and second primary antibody (preferably monoclonal antibody) recesses 13, 14 on the corresponding base 2 and cover 4, wherein the recesses 13 contain dried antibody and the recesses 14 contain suspension. Preferably, at least one wash well 12 (two wells 12 in the example of Figure 1) is interposed between the primary antibody recesses 13, 14 and the first and third recesses 3, 5. Furthermore, the first or third recesses 3, 5 contain primary antibody. Furthermore, the material collection membrane of the recesses carries antigen-specific antibodies of the corresponding primary monoclonal antibody. These antibodies are particularly polyclonal antibodies. Preferably, at least one wash well 12 (two wells 12 in the example of Figure 1) is positioned between the first and third recesses 3, 5 and the second and fourth recesses 3', 5'. Thus, as shown in the figure, the base 2 and cover 4 have an elongated shape so that the recesses and wells can be arranged longitudinally as described above. This also makes it possible to optimize the space required when installing an automated processing machine that can process multiple disposable kits in parallel.

[0058] According to the embodiment shown in the figure, the initial recesses 10, 11 are positioned at the initial positions of a sequence that unfolds longitudinally along the disposable kit 1. According to a preferred embodiment of the present invention, at least one disposable kit 1 is used with a machine that includes a longitudinally movable shuttle, which is positioned beneath the base 2 when in use, and has a lower piercer 16 having material rigidity and / or shape such that when the shuttle reaches the appropriate position it drills into the corresponding bottoms of the recesses 3, 3', 10, 13 and wells 12, 10 (Figure 8). This causes the metered liquid contained in each recess or well, in particular the pre-metered liquid, to flow out by gravity.

[0059] Preferably, the piercer has, for example, a vertically elongated shape, so that the liquid present in the recess and / or well is guided downward by surface tension before drilling with the piercer.

[0060] Furthermore, the shuttle includes a bowl 17 from which the piercer 16 protrudes, receiving liquid that flows in by gravity. The bowl 17 includes a membrane 18 based on, for example, polyvinylidene fluoride or other material used within the sector to support antibodies, on its bottom or other surface which is moistened with a measured amount of liquid, and an unbound antigen-specific polyclonal antibody is locally applied onto this membrane 18 so that it binds to the antigen present in the initial solution containing the biological test material. In the example of Figure 1, the initial solution containing the biological test material, collected by a brush, cotton swab, or site brush 19, is obtained by drilling a first initialization recess 11 and mixing the dry protease inhibitor and dissolving solution with the brush 19. The measured amount of dry inhibitor and dissolving solution, for example 800 microliters of liquid, falls into the bowl 17 by gravity after drilling by moving the piercer 16 upward relative to the base 2.

[0061] The shuttle continues to drill holes in each recess 13 (Figure 8), 3, 5, and well 12 in sequence on the opposite side of the cover 4 relative to the base 2, until varying amounts of liquid accumulate in the bowl 17 until a chemical reaction occurs that produces a bioluminescent effect.

[0062] According to a modified embodiment shown in Figure 7, recesses containing test material based on different primary antibodies for each sequence are preferably arranged in a line on the same disposable kit 1, and in this embodiment, there is also a sequence of reference recesses having the same secondary antibody as the other sequences and one of the primary antibodies of the other sequences. According to a preferred embodiment, the kit comprises a plurality of five recesses 13 containing corresponding preferably monoclonal dried and / or dehydrated primary antibodies (e.g., specific EGFR antibody, specific p53, specific B7H6, specific PdL1, and one of the above antibodies), and a plurality of five recesses 3 containing the same dried and / or dehydrated conjugated secondary antibody, each containing a catalyst for the primary antibody. Similarly, a shuttle transported by a mask M having corresponding wells transports a total of five membranes: four membranes 18 each having a preferably polyclonal antigen-specific antibody combined with a corresponding primary antibody, and a reference membrane 8 having a different antigen from the other four membranes, for example, a preferably polyclonal antibody specific to melanoma antigen. Membranes 8 and 18 are located in the wells of the mask M. Furthermore, the shuttle is equipped with multiple piercers 16 aligned to a corresponding set of five recesses / wells (four recesses for the test material and a fifth recess for the reference material), so that at the shuttle's stopping position, each piercer 16 is beneath the corresponding recesses 13, 3, 3', and during a single relative drilling motion, all of the recesses are drilled and the contained liquid is drained by gravity to the corresponding bowl 17. In the example of Figure 7, both the series and the piercers 16 are positioned along corresponding lines parallel to each other and are superimposed during the relative drilling motion (relative vertical motion). The mask M preferably also mounts the piercers 16, and the shuttle comprises both the mask M, which is wetted with the material to perform the inspection, and a moving support controlled by appropriate actuators to perform the steps up to well detection and acquisition of post-detection digital images by performing the necessary movements in an iterative and programmed manner.

[0063] Preferably, each well 12 and / or first initial recess 10 contains a metered, particularly pre-metered, amount of liquid sufficient to supply the appropriate amount of material to all bowls 17 of the shuttle, and can therefore be drilled by one or at least two piercers 16 during the drilling operation. Preferably, the wells 12 and the first initial recess 10 are arranged along the axis of symmetry of a series of five recesses 13, 3, 3' and 14, 5, 5'.

[0064] Using a kit configured in this way, it is possible to create a kit that leverages both the advantages of bioluminescence, such as higher accuracy and unique interpretation, especially in process automation, and the availability of accurate and usable instruments outside of the analytical laboratory.

[0065] In one embodiment, the kit includes a cytbrush or other instrument for collecting biological material, including cells from the oral cavity. Alternatively, other instruments capable of collecting samples containing oral cells may be used instead of a cytbrush.

[0066] Therefore, the object of the present invention is a kit for the diagnosis and / or prognosis determination of oral squamous cell carcinoma, comprising a signal detection and / or quantification system comprising two series of primary antibodies derived from two different animal species (preferably mouse and rabbit) anti-EGFR, anti-p53, anti-B7H6, and anti-PdL1, and a secondary antibody that conjugates to alkaline phosphatase or peroxidase and targets one of the two animal species from which the primary antibodies are derived, and optionally instructions for use.

[0067] In a further embodiment, the object of the present invention is achieved by a machine for processing the disposable kits described above in accordance with the method described above. The machine comprises a housing for at least one disposable kit, a closing lid movable between an opening for inserting and removing the disposable kit from the housing and a closed position positioned above the kit, a moving shuttle positioned below the housing to support at least one bowl with the membrane and a puncturer for a recess in the base, a mechanism for moving the shuttle and / or puncturer relative to the housing so that the underside of the recess in the base can be drilled when in use, and an electronic control unit programmed to drive the mechanism in accordance with a predetermined sequence of positions of the membrane below the housing.

[0068] This machine is compact, allowing for precise testing to be performed outside of specialized laboratories, such as pharmacies. [Examples]

[0069] This prospective study was conducted from October to December 2022 at the Galliera Hospital (Genoa, Italy), the San Camillo Hospital (Rome, Italy), and the University of Vanvitelli (Naples, Italy).

[0070] All patients who visited the center after receiving a confirmed diagnosis of oral cancer, regardless of age, sex, ethnicity, or overall health status, underwent follow-up examinations.

[0071] All patients enrolled in this study were diagnosed with primary oral squamous cell carcinoma (OSCC) stages I-IV (T1-T4) based on TNM (tumor lymph node metastasis) criteria and had no history of chemotherapy or radiotherapy. All protocols used in this study were approved by the Ethics Review Board and Institutional Review Board of the University of Genoa, Italy, and all participating institutions were registered.

[0072] [Sample collection:] Detailed medical histories were collected using a designated form. Written informed consent was obtained from all patients.

[0073] (Site brush:) Samples for analysis were collected using a site brush (Mercinger SpA).

[0074] Patients were instructed to rinse their mouths with saline solution before the cytobrush biopsy. Three cytobrush samples were collected from each patient's mouth by rapidly rotating the cytobrush 360°, applying sufficient pressure to collect cells and detached tissue while minimizing bleeding. The tips of the cytobrushes were inserted into sealed Eppendorf vials and stored refrigerated until analysis.

[0075] Samples that deteriorated due to failure to follow storage and / or transport protocols, samples containing relevant blood fractions, and samples containing cell fractions below the cutoff threshold (10 femtograms / microliter) were excluded from analysis.

[0076] Three non-invasive cytobrush biopsies were collected from each patient. Each cytobrush was used to rub the target area with light pressure while rotating it. Although the cytobrush was rubbed firmly, care was taken to avoid bleeding from the collection site. Each cytobrush collected cells from one of the three target regions of this study (tumor center, tumor periphery, or healthy control tissue).

[0077] In each patient, one sample was collected from the center of the lesion (Group A), one sample from the periphery of the lesion (Group B), and one sample from surrounding healthy tissue (Group C). The samples were listed, refrigerated at 0-4°C, and sent to the laboratory in a refrigerated container for analysis.

[0078] (Biopsy:) Biopsy tissue samples (one for the tumor center, one for the tumor periphery, and one for healthy tissue) were placed in a sealed refrigerated container (0-4°C) and washed three times with 1 mL of PBS (Dulbecc's SERVA buffer) using a rocking shaker (ARGO-LAB SKO-D XL). The tissue samples were cut to approximately 1 square centimeter in size, placed in a 2 mL Eppendorf filter, and 800 μL of lysis buffer (EMD millipore RIPA lysis buffer) and a protease inhibitor cocktail (SIGMA Protease inhibitor cocktail) were added. The solution was homogenized for 10 minutes using an automated homogenizer (PREOMICS BeatBox), and then centrifuged at 2000 r for 10 minutes (Healttrow scientific SPROUT PLUS). The supernatant was used for subsequent analysis.

[0079] (Analysis:) Samples were processed using the automated instrument Femtohunter (manufactured by Stark Sarl, Principality of Monaco). Femtohunter is an instrument that enables rapid quantitative ELISA testing using chemiluminescence. When using the kit of the present invention, this system has an analytical sensitivity of 10 femtograms / microliter.

[0080] (Sample analysis:) Biological samples were inserted into the corresponding cavities in the reagent slots of the Stark kit.

[0081] Marker analysis was performed in the following order: The first analysis slot consisted of EGFR, p53, and Ki67 markers and a control membrane, while the second analysis slot consisted of B7H6, PDL1, and HLAE markers and a control.

[0082] The membrane is a PVDF membrane (Thermofisher Scientific catalog number LC2002).

[0083] A reagent slot containing a patient-derived biological sample was inserted into an automated dispensing device equipped with a chemiluminescent ELISA process.

[0084] A slot membrane loaded with a polyclonal antibody against the target marker was inserted into an automated dispensing device. The automated dispensing process consists of 12 steps for the ELISA process and 1 step for optical signal detection and analysis, for a total of 13 steps.

[0085] 1. Activation of the PVDF membrane 2. Load the dissolved biological sample into the membrane slot. 3. Clean the membrane and remove any excess unbound material. 4. Add BSA solution to block nonspecific sites on the PVDF membrane. 5. Wash the membrane and remove unbound proteins. 6. A monoclonal antibody against the target marker is loaded onto a PVDF membrane to form a sandwich structure. 7. Clean the membrane and remove any nonspecific deposits. 8. Clean the membrane and remove any nonspecific deposits. 9. Load the HRP-labeled secondary monoclonal antibody onto the membrane. 10. Clean the membrane and remove any nonspecific deposits. 11. Clean the membrane and remove any nonspecific deposits. 12. Load the chemiluminescent sensing substrate onto the membrane. 13. Detect the chemiluminescence signal and analyze the light intensity present on the PVDF membrane at a wavelength of 425 nm.

[0086] (antibody:) Polyclonal antibody anti-EGFR protein GENETEX GTX121919, monoclonal antibody anti-EGFR protein GENETEX GTX628887, polyclonal antibody anti-AR protein GENETEX GTX100056, monoclonal antibody anti-AR protein ABCAM ab9474, polyclonal antibody anti-ER protein GENETEX GTX127978, monoclonal antibody anti-ER protein GENETEX GTX70171, polyclonal antibody anti-p16R protein ABCAM Ab189034, monoclonal antibody anti-p16 protein ABCAM Ab201980, polyclonal antibody anti-p53 protein ABCAM Ab131442, monoclonal antibody anti-p53 protein ABCAM PAb1801, polyclonal antibody anti-Ki67 protein ABCAM Ab15580, monoclonal antibody anti-Ki67 protein INVITROGEN MA5-15690, polyclonal antibody anti-PDL1 protein ABCAM Ab233482, Monoclonal antibody anti-PDL1 protein R&D SYSTEMS MAB1561R, Polyclonal antibody anti-B7H6 protein ABCAM ab229999, Monoclonal antibody anti-B7H6 protein R&D SYSTEMS MAB7144, Polyclonal antibody anti-HLAE protein ABCAM Ab203082, Monoclonal antibody anti-HLAE protein Antibodies.com A121801.

[0087] (Statistical analysis:) All statistical analyses were performed using statistical software R2.9.1 and Bioconductor 2.5 (packages: ROC, meta). For samples from OSCC test groups A, B, and control group C, the mean and standard deviation of each marker were calculated. Differential expression of each marker was assessed within each cohort by the Mann-Whitney U test to determine statistical significance and by constructing receiver operating characteristic curves (ROCs). The area under the curve (AUC) of each ROC curve was obtained by numerical integration. Sensitivity and specificity were assessed for each marker in each group using the expression cut-off point corresponding to the p-th percentile value of the marker, where p is the proportion of OSCC cases in the studied cohort.

[0088] In the meta-analysis of individual markers, sensitivity and specificity estimates were used, and pooled estimates were generated using inverse variance meta-analysis models with fixed and random effects. These models were constructed separately for sensitivity and specificity and weighted proportionally to the relevant sample size of each study. Due to differences in measurement scales between experiments and differences in the proportion of OSCC cases in each cohort, sensitivity and specificity for each cohort were used instead of the original marker values. A meta-analysis comparing marker expression between cancer and control was also performed. In this analysis, expression levels between cohorts were normalized using within-group Z-transformation, and then the Z-transformed values ​​between cancer and control were compared using a mixed-effects model. In the evaluation of classification models, logistic regression was used to examine the usefulness of marker combinations in the classification of OSCC. Two models were constructed for each cohort. First, the same markers as in the original report were used in each group. Second, stepwise advanced regression was used within each group to identify the top three markers in each group. AUC was calculated by constructing ROC curves using the probabilities predicted by each logistic model and integrating them numerically. The sensitivity and specificity of the model were calculated in the same way as for the individual marker models.

Claims

1. An in vitro method for the diagnosis and / or prognosis determination of oral squamous cell carcinoma, comprising the following basic steps: A step of preparing a sample isolated from the subject, Steps to analyze the protein levels of EGFR, p53, B7H6, and PdL1 in the aforementioned sample. The step of comparing the aforementioned level with the level of the corresponding protein in a sample isolated from a control group, and / or the level recorded in a reference database, Here, the change in terms of an increase in the level of the protein in the sample indicates the presence of oral squamous cell carcinoma in the subject.

2. The method according to claim 1, wherein the step of analyzing the protein level is performed using the ELISA method.

3. The method according to claim 1 or 2, wherein the sample is isolated from any sample including a buccal mucosa swab, a cytological smear, saliva, or oral epithelial cells, or from a biopsy including oral epithelial tissue.

4. The method according to any one of claims 1 to 3, wherein the analysis step includes a step of detecting via bioluminescence and a step of detecting a digital image of the detection and processing of the bioluminescence, preferably by binarizing the image to associate each image pixel with a background label corresponding to a lower brightness or an activated pixel label representing a bioluminescent effect and corresponding to a higher brightness.

5. A kit for the diagnosis and / or prognosis determination of oral squamous cell carcinoma, An instrument suitable for collecting oral samples containing cells, preferably a site brush, Two series of primary antibodies derived from two different animal species, preferably one series of monoclonal antibodies and one series of polyclonal antibodies, anti-EGFR, anti-p53, anti-B7H6, anti-PdL1 E'MEGLIO ANTI o AGAINST? and, A signal detection and / or quantification system comprising a secondary antibody conjugated to a detection compound, preferably alkaline phosphatase or peroxidase, and comprising a secondary antibody that targets one of two animal species from which the primary antibody originates, A kit that includes optional user instructions.

6. The kit according to claim 5, wherein the kit is disposable, and two sets of primary antibodies are present, one immobilized on a support, for example, a solid support of PVDF, and the other dried / freeze-dried so as to be solubilized upon use.

7. A disposable kit according to claim 6, comprising a base (2) having a first plurality of recesses (13) for one of a series of primary antibodies, and a cover (4) having a second plurality of recesses (14) for a primary antibody solubilizer, wherein when the cover (4) is placed on the base (2), the second plurality of recesses (14) are housed within the first plurality of recesses (13), and at least the second plurality of recesses are perforated by hand using a tool.

8. The disposable kit according to claim 7, wherein at least one recess (13, 14) is closed or sealed at the top by a peelable or perforated layer before use.

9. The disposable kit according to any one of claims 6 to 8, further comprising a disposable mask (M) having at least one recess on which the other of the two sets of primary antibodies is immobilized.

10. Use of the kit according to any one of claims 5 to 9 in an in vitro method for the diagnosis and / or prognosis determination of oral squamous cell carcinoma.

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