A METHOD AND KIT FOR THE DETECTION OF A BIOMARKER OF THYROID CANCER IN BIOLOGICAL SAMPLES

DESCRIPTION

Field of the Invention

The present invention relates in general to the field of medical biotechnology, and more particularly it refers to a method and related kit for the detection of a biomarker associated to thyroid tumours in biological samples.

State of the Art

Thyroid nodules are found very frequently in the clinical practice: even in areas not deficient in iodine, they are observed with an incidence of 4-7% in the adult population. By contrast, with the exception of the areas that have suffered a nuclear fallout as a result of a nuclear accident, the vast majority of the diagnosed thyroid nodules are then discovered to be benign lesions and the incidence of malignant thyroid tumours is relatively low: between 1.2 and 2.6 cases of thyroid cancer have been reported per 100,000 men with nodules, and these numbers rise up to between 2.0 and 3.8 for the same number of women with thyroid nodules, with a higher incidence in some countries, such as Sweden, France, Japan and the United States (see for instance Siegel R. et al. "Cancer Statistics" in 2012, CA Cancer J. Clin. 62: 10- 29).

While medical therapy for thyroid cancer involves the surgical treatment until complete removal of the thyroid or "thyroidectomy", in the case of benign nodules the treatment is limited to a clinical and echographic follow-up without any surgical treatments, except of course in cases where the nodule is so large as to cause compression on nearby organs with consequent difficult swallowing and/or breathing. This is why a proper diagnosis of the benign or malignant nature of the thyroid nodules has an even greater importance than in other types of pathologic conditions, in order to avoid not needed surgery and associated risks for the patient, subsequent treatments to restore hormonal balances, medical and health expenses, etc.

The diagnosis of thyroid nodules, which allows to discern between benign and malignant tumours, is currently carried out with the well-established technique of fine- needle aspiration cytology (or FNA), wherein the cellular material taken from the patient with the needle aspiration technique is examined under a microscope. This technique involves the aspiration of the material with a very fine needle that is driven into the thyroid nodule to be examined.

This procedure, while being relatively simple and consolidated, has however a certain degree of invasiveness and provides a series of manoeuvres on the patient that must be necessarily performed by a physician experienced in endocrinology, or by a suitably trained cytopathologist. The risks for the patient, for instance to undergo bleeding, are not null and the chances of having to repeat several times the examination because of inadequacy of the aspirated material, are high. Moreover, the cytological examination does not allow to discern between the microfollicular neoformations, that would not require thyroidectomy, and the malignant tumour lesions, with the result that even in the case of a microfollicular lesion a surgical treatment is however performed, and with all the consequences that this involves in terms of the risks linked to the surgical operation and in terms of the need for a drug treatment to compensate for the lack of production of thyroid hormones and, not least, in terms of healthcare costs.

It is therefore much felt in the field the need of having available a diagnostic kit and related method for the correct diagnosis of the thyroid tumours, which allows discerning with certainty between benign and malignant thyroid nodules under investigation, and further identifying microfollicular nodules, without necessarily using the fine needle aspiration.

Summary of the Invention

Now inventors have performed a comparative proteomic analysis of a number of samples of biological material collected by needle aspiration, by comparing the protein patterns derived from patients with different types of thyroid cancer, with the patterns obtained from patients with benign lesions or with microfollicular lesions. Thanks to these studies the inventors have identified a biomarker that has a high sensitivity and specificity in discerning between benign and malignant lesions, and they have developed a method and a kit for the assay of this biomarker in an extremely sensitive and specific way, which represents a very rapid and non-invasive system, able to support the physician in the diagnosis of thyroid tumours and to discern with certainty between benign and malignant forms, also assisting in distinguishing the microfollicular lesions.

Besides being supportive to the physician in making a correct diagnosis, the kit and method of the invention allow to make the diagnosis on samples of saliva or serum of the patient under examination, with practically no invasiveness and much lower invasiveness with respect to the traditional examination of fine needle aspiration, even if the material obtained from a fine needle aspiration can still be used as a biological sample in the method of the invention, for instance for further consideration and confirmation of a diagnosis carried out with the traditional method of the cytological analysis of the sample from fine needle aspiration.

It is therefore subject of the invention a method for detecting the presence and/or determining the concentration of annexin 1 in a biological sample, whose essential features are defined in the first of the attached claims.

A kit for the detection and/or the quantification of the protein annexin 1 in a biological sample, whose essential features are defined in the attached claim 7, is a further subject of the invention.

Characteristics and advantages of the method and kit according to the present invention will result clearer from the following detailed description of an exemplary and non-limiting embodiment thereof.

Brief description of the drawings

Figure 1a: histograms of the mean values of intensity of the signal obtained with the ELISA tests for the detection of anti-annexin 1 antibodies, described in the following experimental part, in samples of a material from fine needle aspiration in a B group of control patients suffering from benign thyroid nodules and in a group C of patients with malignant thyroid nodules.

Figure 1b: as in Figure 1a in serum samples instead of in material from fine needle aspiration.

Figure 1c: as in Figure 1a in saliva samples instead of in material from fine needle aspiration.

Detailed Description of the Invention The inventors have performed studies and identified the protein annexin 1 as a biochemical marker present in different types of biological samples, especially in blood samples, in the saliva and in samples from needle biopsy, also finding that it is extremely specific to the presence of malignant tumours of the thyroid.

According to a particularly advantageous embodiment of the present invention the biological samples are saliva samples that allow accomplishing the present method with no invasiveness for the patient, together with an extreme simplicity and quickness in the sampling procedure and the preparation of the sample.

The presence of the above said serious diseases can therefore be detected by searching this biomarker, and verifying the levels, in samples of patients who have the need for a diagnosis in this respect, for instance in patients having thyroid nodules.

The method of the invention is therefore a method for detecting the presence and/or measuring the concentration of annexin 1 in a human biological sample, comprising the following steps:

a) preparation of said biological sample previously obtained, consisting of a blood sample, a sample of saliva or of a material from needle biopsy;

b) determination of the presence and/or measuring of the concentration values of annexin 1 in said biological sample;

c) possible comparison of the concentration values obtained from step b) with reference values;

d) association of the presence of annexin 1 and of concentration values obtained to a pathological situation that is a thyroid tumour.

According to the present invention by "blood sample" is meant a sample of serum, plasma or whole blood, optionally added with substances commonly used in the clinical analyses, for instance anti-coagulant substances. Preferably by "blood sample" the serum is meant.

By "thyroid tumour" is meant a malignant tumour of the thyroid, for instance a thyroid malignant lymphoma, a thyroid papillary cancer, a thyroid medullary cancer, a thyroid anaplastic carcinoma or a metastatic carcinoma of the thyroid.

The detection of the presence of annexin 1 and the determination of the related concentration values in a biological sample is carried out according to the invention by an immunological assay using anti-annexin 1 monoclonal, polyclonal or recombinant antibodies, preferably monoclonal antibodies that recognise epitopes of the sequence of annexin 1 comprising the fragment 219-267. The annexin 1 is identified by the aminoacid sequence SwissProt Acc. No. P04083. According to a particular embodiment of the invention commercial anti-annexin 1 antibodies, such as the monoclonal antibody #8691 (Annexin A1 (D16A10) Rabbit mAb) by Cell Signaling Technology Inc., may be also used.

The immunoassay used in the method of the invention is preferably a direct ELISA assay of the type so-called "sandwich"; by ELISA assay is meant the well-known assay which takes its name from the English "Enzyme-Linked Immunosorbent Assay," or immunosorbent assay linked to an enzyme, and that is used to detect the presence of an antigen by use of one or more antibodies, one of which is linked to an enzyme.

In the direct sandwich ELISA test, usually carried out in microtitre plates provided with wells, the bottom of the wells is coated with a specific antibody to be detected (primary antibody), then, after at least one washing, it is added the sample in which the presence of the antigen is to be detected, optionally also measuring the antigen concentration; it is then incubated for the time required for the antigen to bind to the antibody, and then at least one further washing is performed to eliminate the antigens in excess. At this point a second specific antibody (secondary antibody) is added in the well, which was capable of binding the antibody-antigen complex possibly present, thus obtaining a kind of sandwich, from which the assay just takes its name. To the second specific antibody added is bound an enzyme that allows the chromogenic detection of the antigen: indeed, after further washings, by addition in the well of the substrate of the enzyme linked to the second antibody, a coloured product formed. The colour development indicates the presence of the antigen and, by means of spectrophotometric measurement of the colour; it is possible to determine the concentration of the antigen present in the sample.

The colour development may also be obtained by directly binding the antibody to a chromophore substance.

The method according to the invention allows therefore performing a diagnosis on patients having thyroid nodules on blood or saliva samples. The method of the invention may also be used for confirming an already proposed diagnosis based on the results of a fine needle aspiration analysis, by using blood or saliva samples, or even on samples of the same material coming from needle aspiration. The diagnosis with the method of the invention allows detecting the presence of a thyroid malignant tumour in case of detection of the protein annexin 1 in the sample under examination. The method of the invention is extremely sensitive and allows detecting even minimum amounts of annexin 1 in a biological sample, typically up to amounts lower than a nanogram per millilitre of sample, for instance amounts of 0.78 ng/ml of sample, allowing for instance to discern with reasonable plausibility between malignant and benign thyroid nodules, as well as to associate the presence of a follicular lesion to specific concentration values measured for annexin 1.

A further subject of the invention is a kit for the detection and/or the quantification of annexin 1 in a human biological sample that is a blood sample, a saliva sample or a sample of a material from fine needle aspiration for the identification of thyroid cancers in patients in need of such a diagnosis, for instance in patients having thyroid nodules. The kit of the invention comprises at least an antibody capable of recognising the annexin 1 , and preferably an epitope thereof comprising the fragment 219-267 (SEQ. ID. N. 1 ) of the sequence of annexin 1.

According to a preferred embodiment of the present invention, the above said kit comprises at least two antibodies for carrying out a sandwich ELISA assay, a primary antibody specific for annexin 1 and a secondary antibody able to bind an immunocomplex between said primary antibody and the annexin 1. Preferably, the secondary antibody is linked to an enzyme that in the presence of a certain substrate, causes a chromogenic reaction wherein the production of colour is directly proportional to the amount of annexin 1 linked in the immunocomplex; by means of spectrophotometric analysis of the solution, it is therefore possible to determine the concentration of annexin 1 present, which can be so precisely dosed.

Experimental Part

In the following experiments samples of saliva, serum and material from fine needle aspiration were used, which were taken from patients having thyroid nodules. The patients gave their written consent, free and informed, to the sampling and the study carried out on the material taken, and described herein below, as also approved by the local Ethics Committee of the Azienda Ospedaliera Pisana.

Collection and preparation of the samples

Fine needle aspiration samples

The material from fine needle aspiration used in the present experiments is not the material from fine needle aspiration traditionally used for diagnostic purposes by conventional cytological analysis, but in the present case, in an echo-guided pre- surgical procedure of fine needle aspiration (FNAB, Fine Needle Aspiration Biopsy), after the sampling of material for the cytological analysis, it was used the portion of material left in the needle, recovered by washing with 300 μΙ of physiological saline solution and centrifugation at 2300 xg for 10 minutes at 4°C. The supernatants were stored at the temperature of -80°C and subsequently used for the experiments.

Saliva samples

The saliva samples used here were collected in the early morning from patients under standard conditions, i.e. on an empty stomach, without taking food or drinks from the evening before. To minimize protein degradation, the samples were immediately processed and maintained on ice during processing. From each subject were collected between 1 and 3 ml of saliva. To remove debris and cells, a centrifugation was carried out at 14,000 g for 30 minutes at 4°C; the supernatants were recovered and stored at - 80°C until use.

Blood samples

Blood samples have been taken from patients under the standard conditions said above, and centrifuged at 3500 g per 10 minutes at room temperature. Serum samples obtained are collected and stored at -80°C.

Preparation of the microtitre plate with anti-annexin 1 antibody

A polystyrene microtitre plate with wells was used, which was coated with the anti- annexin 1 antibody, incubated for 48 hours at room temperature. A specific antibody anti- annexin 1 was selected that was able to recognize a sequence with low homology compared to other annexins. An antibody specific to annexin 1 may be obtained against the sequence 219-267 (NVFNTILTTRSYPQLRRVFQKYT YSKHDMNKVLDLELK GDIEKCLTAI; SEQ ID. N. 1 ), it was therefore selected an antibody against a region around Val 237 of the sequence of annexin 1 , that is marketed by Cell Signalling Technology Inc. under the name #8691 Annexin A1 (D16A10) Rabbit mAb.

The antibody was diluted up to a final concentration of 1 μg/ml in carbonate buffer (Na ₂ C0 ₃ 25 mM, NaHC0 ₃ 25 mM, pH 9.7) before being used for covering the wells of the microtitre plate, as described above.

After washing with T-PBS, that is phosphate-buffered saline and Tween ^® in the following proportions NaH ₂ P0 ₄ 20 mM, NaCI 0,9%, pH 7.15 and Tween ^® 0.05%, the binding sites of the protein remaining in the coated wells of the microtitre plate were blocked by adding a blocking buffer, consisting of 3% of bovine serum albumin in T-PBS, and leaving in incubation for 2 hours at room temperature.

Creation of the calibration curve

The calibration curve was obtained by using standard purified annexin 1 , heated at 100°C for 5 minutes, then subjected to serial dilutions in PBS-T with detergent solution (0.06% of zwitterionic detergent CHAPS and urea 150 mM) until the following final concentrations of standard annexin 1 are obtained, expressed in ng/ml: 50, 25, 12.5, 6.25, 3.12, 1.56 and 0.78.

Incubation of the samples

The wells were washed once with T-PBS and incubated with the samples that were treated in a different way depending on the type of sample, from fine needle aspiration, from saliva or blood of the patient.

Before being added to the microtitre plate's well, the samples from fine needle aspiration were diluted 1 :20 in T-PBS and heated for 5 minutes to 100°C; after cooling, the detergent was added up to a final concentration of zwitterionic detergent CHAPS of 0.3% and of urea of 750 mM. The samples obtained from saliva were diluted 1 :500 in T- PBS and heated to 100°C for 5 minutes; after cooling, the detergent was added up to a final concentration of zwitterionic detergent CHAPS of 0.06% and of urea of 150 mM. The serum samples obtained from blood sampling of patients were diluted 1 :100 in T- PBS containing the zwitterionic detergent CHAPS with a final concentration of 0.06%, then they were heated to 100°C for 5 minutes; after cooling, urea up to a final concentration of 150 mM and dithiothreitol up to a final concentration of 60 mM, were added. Primary and secondary antibodies

After incubation for 2 hours at 37°C the plate was washed once with T-PBS and the primary antibody anti-annexin 1 was added, a mouse full-length antibody different from the antibody used for coating the wells, at a concentration of 0.5 μg/ml.

After incubation for 1 hour at 37°C, the plate was washed for 5 times with T-PBS and then incubated for 30 minutes, at 37°C, with the secondary antibody anti mouse Perkin Elmer for forming the immune complex.

Detection

After five washings with T-PBS, in each well aliquots of 100 μΙ of the solution of chromogenic substrate 3,3',5,5'-tetramethylbenzidine (TMB) have been added and, after further 20 minutes at 37°C for the colorimetric reaction to occur, the blocking solution of sulphuric acid 0.3-0.5 M was added in the plate's wells, then reading the optical absorbance at 450 nm in each well by means of a reader suitable for microtitre plates. The concentration of anti-annexin 1 antibodies in each patients under examination was derived from the so-measured absorbance values, thus finding a sharp increase in the concentration of anti-annexin 1 antibodies in the group of patients with malignant thyroid nodules compared to group of control patients, suffering from benign thyroid nodules.

In Figures 1a, 1b and 1c are illustrated the results obtained respectively for the fine needle aspiration samples, serum samples, and saliva samples, in each case for both the patients in the control group (group B) and the patients suffering from thyroid malignant tumour (group C). Each histogram in the Figures represents the mean value M ± SEM (Standard Error of the Mean) of the signal intensity, wherein ^* indicates p<0.05 and ^** indicates p<0.01.

The present invention has hereto been described with reference to a preferred embodiment. It is to be understood that there may be other embodiments of the same invention, as defined by the scope of protection of the claims set out below.