MX2010005476A - Diagnosis method for the early detection of cervicouterine cancer by detecting the eag1 protein as a marker of cervical intraepithelial lesions. - Google Patents

Abstract

The present invention describes a method for the early detection of cervicouterine cancer (CuCa) by detecting the human protein Eag1 with specific antibodies. The Eag1 protein is differentially expressed in cervical tissue and in close association with the stage development of the disease, thereby cancer being detected in early phases, thus the aforementioned method may be useful as an adjuvant in the opportune detection of CuCa. In addition, the inventive method intends to reduce the percentage of false positive or negative obtained in the cytological detection test (Papanicolaou) and may increase the detection sensibility in the tests currently used for the diagnosis of CuCa.

Description

Diagnostic method for the early detection of cervical cancer by detecting the Eagl protein as a marker of cervical intraepithelial lesions Field of the invention.

The present invention is related to the development of diagnostic methods for the early detection of diseases, more particularly with diagnostic methods for the timely detection of human cervical uterine cancer from early stages through the detection of the Eagl protein.

BACKGROUND OF THE INVENTION Cervical cancer (CaCu) is considered a health problem in developing countries, so the detection of precancerous lesions in the cervix is one of the main issues to be resolved in health programs.

It is estimated that the CaCu causes around 500,000 deaths per year in the world; It is the most common cancer of women in developing countries, where it is present with an incidence of up to 40 per 100,000 women1. For example in Mexico, CaCu represents 21.5% of all cancers detected among the population and 33.2% of cancers detected in women, being the main cause of cancer death in women, with a mortality rate of 14 for every 100,000 women over 15 years of age. The majority of cases are observed between 25 and 54 years of age, with a maximum peak between 30 and 492.

It has been shown that CaCu is preceded by an ordered series of epithelial changes, which microscopically range from cellular atypia to various degrees of dysplasia (cervical intraepithelial neoplasia, CIN), which ultimately lead to the development of cancer. Precancerous lesions occur approximately 10 to 15 years earlier than invasive cancer. However, it has been observed that not all dysplasias progress to invasive carcinoma. It is estimated that only 5-15% of severe NICs suffer a progression to invasive cancer in a period of 3 years; while the progression in mild NICs is only 1% In the etiology of CaCu and its precursor lesions, several viral agents have been linked, such as human papilloma virus (HPV), herpes simplex virus and human immunodeficiency virus. By means of molecular techniques, it has been established that HPV is directly related to the process of cervical carcinogenesis3,4, which virus has been found in up to 99% of invading cancers and from 70 to 80% in preinvasive lesions. Although this infection is considered the main cause of CaCu, the majority of infections caused by HPV usually pass asymptomatic5,6.

The cellular morphological changes found in the cervix are called cervical dysplasia or Cervical Intraepithelial Neoplasia (CIN). The different degrees of dysplasia are located only in the squamous epithelium and morphologically depend on their presence in the lower, middle or full thickness third.

The squamous intraepithelial lesions (LEI) group a series of epithelial abnormalities that can range from transient changes caused by HPV to precursor changes of an invasive lesion; These changes are divided into low (LEI-b) and high-grade (LEI-a) injuries. LIE-b include cytopathic changes produced by HPV infection called koilocytosis, moderate dysplasia, or CIN1; while the LIE-a include severe dysplasias and carcinoma in situ, previously called CIN2 and CIN3.

The risk of developing an IAS is proportional to the number of HPV-positive specimens, suggesting that carcinogenic development results from persistent infections. It has recently been established that the main cause of CaCu is HPV infection; in this sense, using the PCR technique in samples from patients with CaCu it has been shown that HPV DNA is present in most cases (table 1), a fact that suggests that HPV infection could be a necessary cause for the development of CaCu9.

Table 1 Correlation of HPV detection with cytology LEI Squamous intraepithelial lesions.

ASC-US Atypical squamous cells of undetermined significance.

Likewise, epidemiological studies on CaCu identify HPV as the main risk factor for its development6. In fact, prophylactic vaccines against HPV have been shown to be a significant protection against their infection and thus decrease the incidence of CaCu10,11. Additionally, estrogens are also considered a factor that contributes to the development of this type of cancer, since the prolonged use of oral contraceptives containing estrogen increases the risk of this disease12'13. In this case, estrogens are involved in the proliferation of various cancer cells and in the expression of estrogen receptors (ER) or aromatase, demonstrating that these molecules stimulate the proliferation of tumor cells derived from mammary gland, lung and CaCu14,15 , 16.

The detection of CaCu is traditionally performed using the technique established by Papanicolaou, whose efficacy is due to the accessibility for the intake and the long preclinical period of the disease in which it is detectable. The application of detection programs throughout several countries using this technique has significantly reduced the mortality due to CaCu. Through various studies, it is suggested that the probability of having invasive cancer of the cervix decreases by 90% if the study of detection is done every 3 years; although the guidelines of expert groups for the detection of CaCu continue to recommend the annual study, decreasing the frequency to 3 years after 3 negative studies17.

However, despite the successful introduction of the Pap test and the existence of several tests (for example, colposcopy) to detect precancerous lesions in cervical tissue, CaCu remains an important public health problem in many countries, since especially in developing countries, CaCu is one of the main causes of cancer mortality among women1,18. One of the main reasons for this effect is that cervical cytology presents important limitations due to the high proportion of false negatives that occur, since about one third of the results can be attributed to errors in the interpretation by the cytopathologist while the remaining two thirds are due to an error in the taking of the sample and preparation of the plate to be examined19; Due to these effects, cytological tests have a heterogeneous sensitivity range that varies from 30 to 87%. On the other hand, the test lends itself to subjective criteria of interpretation and requires laboratories with highly qualified personnel; In this sense, more reproducible tests are needed to improve current detection tests to avoid unnecessary medical intervention and very importantly, to identify cases that may go unnoticed with current methods.

Due to the great uncertainty that the Papanicolaou test provides, several methodologies have been developed so far to try to eliminate the problems associated with it, which include the detection of various molecules associated with the development of the disease.

For example, methods have been described to detect and identify potential biomarkers of high-grade cervical dysplasia in patients, 20 or the use of intracellular CV1 as a biomarker21.

On the other hand, for the early detection of CaCu, methodologies have been reported that involve the detection of alterations of the MAL22 gene, the detection of the human protein of fibroblast activation (FAP / seprase) 23, as well as the detection of the HCC gene 24; Likewise, methodologies that use specific antibodies directed to certain biomarker molecules have been reported., such as the cell proliferation marker PCNA25, the peptides E2, E6 or E7 of the early coding region of HPV26, proteins of HPV27, HLA-DR antigen of the major histocompatibility complex28 and the E7 protein of HPV29. However, the previous methodologies become complex in their implementation and interpretation because it is necessary to use several molecules or antibodies at the same time to achieve the detection of the molecules chosen as biomarkers in CaCu.; For example, in the case of methodologies that use antibodies to detect HPV proteins, it is necessary to use various antibodies that specifically recognize the different types of HPV associated with the development of CaCu.

Clearly, HPV infection and the use of estrogens are considered recognized risk factors in the development of carcinogenesis of the cervix, however recently the appearance of some ion channels such as Eagl potassium channels in cervical tissue have taken on particular importance as possible Risk markers for the development of cervical cancer.

Despite the restricted expression of Eagl potassium channels in normal tissues, these channels have been identified in several malignancies, including cervical cancer30,31. Additionally, previous reports have shown that in some samples from patients with a cytologic diagnosis negative for cancer, including various gynecological pathologies such as HPV infection, a high expression of Eagl mRNA has been found, suggesting its possible use in the detection of cancer. cervical30.

The channels of potassium ether a go-go 1 (Eagl) have oncogenic properties32; The human Eagl mRNA shows a restricted distribution in healthy tissues, it is expressed mainly in the brain, a little in the placenta, the testicles, the adrenal gland and transiently in the myoblasts, just before the fusion of the cell31'33. In contrast, the gene and the eagl protein are more abundantly expressed in cells Cancer of different histogenesis including CaCu, lung cancer, breast cancer, colon cancer and in prostate cancer30,31.

Different investigations have considered the Eagl channel as a powerful tumor marker for many types of cancer, since they have demonstrated their participation in the development of cancer. For example, some findings suggest the association of the eagl gene amplification with reduced survival in patients with colon carcinoma, 34 while the inhibition of the expression of the eagl gene by antisense oligonucleotides or the silencing of RIMA, significantly decreases the proliferation of several tumor cells32'35,36. Likewise, the inhibition of Eagl channel activity by non-specific inhibitors of Eagl such as imipramine or astemizole has been suggested to decrease the proliferation of cancer cells35'36,37. Likewise, specific blocking of Eagl with monoclonal antibodies inhibits the growth of tumor cells in vitro and in vivo38. Due to the above, Eagl is also considered as a promising membrane target in cancer therapy.

Regarding the possible relationship that Eagl maintains as a tumor marker in the CaCu, Farías and collaborators30 reported that some samples of cervical tissues from female patients diagnosed as normal due to a negative result by Papanicolau smear, showed the expression of Eagl mRNA.; In this case, one of the patients presented infection with HPV, another presented a paratubaric cystadenoma, and a third presented endometrial hyperplasia. These findings suggest that Eagl could be an early potential indicator of cell proliferation, in addition to the fact that Eagl mRNA has been detected in breast tumor tissue of mammary gland "tumor free" 31 and in human diverticulitis, which has the potential of becoming colon cancer34. All these observations suggest that Eagl expression is an early sign of possible cellular hyperproliferation, which is consistent with the fact that estrogen and HPV oncogenes (E6 / E7) regulate the high gene expression of the gene and the eagl protein. in normal cells and / or in cancer cells39. In addition, imipramine, astemizole and monoclonal antibodies that inhibit the activity of the Eagl channel, decrease cell proliferation of keratinocytes transfected with oncogenes E6 / E739. This regulation of Eagl by etiological factors of CaCu strongly suggests that Eagl can be detected in precancerous lesions of the cervix.

Therefore, it is necessary to have new methods of timely detection of CaCu from its early stages of development that include new markers and indicators of CaCu directly related to the early development of the disease, for example Eagl, and without presenting the problems of interpretation and implementation of the CaCu diagnostic and detection procedures developed so far.

Objectives of the invention.

It is one of the objects of the present invention to provide a method for detecting CaCu from early stages of the disease by detecting the Eagl protein in cervical uterine tissue.

Another objective of the invention is to provide useful molecular biomarkers, such as the Eagl protein, for the early detection of CaCu from early stages of the disease.

Another objective of the invention is to provide a method for detecting CaCu from early stages of the disease by detecting the Eagl protein in cervical tissue through the use of specific antibodies against said protein.

Another objective of the invention is to provide a highly sensitive method for detecting CaCu from early stages of the disease by detecting the Eagl protein in cervical uterine tissue.

Another objective of the present invention is to provide a method for detecting the presence of the Eagl protein as a possible risk indicator of developing intraepithelial lesions in cervical tissue of patients who consume estrogen either as part of a contraceptive treatment in reproductive stage or as part of a hormone replacement therapy in menopause.

Brief description of the figures.

Figure 1. A scheme of the grouping of the cervical tissue samples analyzed in the present invention is shown.

Figure 2. The expression of the Eagl protein in various samples is shown as positive and negative controls for ICQ and IHQ. It is observed (A) the expression of the Eagl protein in the soma of neurons (black arrows) of healthy human hippocampus and in dendritic and axon extensions (open arrow), (B) the expression of Eagl in trophoblast (black arrows) and in vascular endothelium (open arrows) of healthy human placenta, (C) expression of Eagl in primary culture cells of CaCu from the biopsy of a patient; the mark is observed in the cytoplasm of normal tissues (placenta and hippocampus) and. mainly in the nucleus (black arrows) of the cells of the primary culture of CaCu, (D) expression of Eagl in biopsy of LIE-BG positive to HPV (the positivity is observed mainly in basal and intermediate cells, (E) negative control in human placenta and (F) negative control in primary culture cells of CaCu.

Figure 3. The expression of Eagl in HPV positive cells is shown. They are observed (A) normal keratinocytes, negative to HPV and Eagl, (B) keratinocytes that express the oncogene E6, (C) keratinocytes that express the oncogenes E6 / E7; the internal boxes show the negative controls in the same cells in the absence of the primary antibody for Eagl, (D) HeLa cells (HPV16 positive), (E) SiHa cells (HPV18 positive); Eagl expression is observed mainly at the nuclear level (black arrows) and (F) negative control in SiHa cells in the presence of the fusion protein.

Figure 4. The expression of Eagl in cervical cytologies is shown. It is observed (A) cytology without evidence of intraepithelial lesion and negative for Eagl, (B) intermediate cells positive in the nuclear membrane (black arrow) and cytoplasm (open arrow) in sample without evidence of cervical intraepithelial lesion, (C) injury LIE-BG, where a positive coilocyte is observed for Eagl in the nuclear membrane (black arrow) and cytoplasm (open arrow) and (D) LIE-AG lesion, where cells with an expression extended to all subcellular compartments are observed.

Figure 5. Eagl expression is shown in morphologically normal cells from LIE. We observed (A and B) positive intermediate cells of a cytology with LIE-BG, and (C) intermediate cells positive of a cytology with LIE-AG; in both cases the positivity was mainly detected in the nucleus (black arrows) and cytoplasm.

Figure 6. An ICQ assay for HPV and p6 is shown. It is observed (A) positive cervical cytology to HPV (coilocyte), (B) positive cervical cytology for p6, (C) cervix biopsy with HPV infection and (D) positive HeLa cells for p6.

Detailed description of the invention.

Considering the reported oncogenic potential for the Eagl protein, as well as the presence of currents, mRNA and protein in various human cancer cell lines and tissues, its lack of expression in most healthy tissues and particularly its recent demonstration in cervical tissue biopsies that were initially diagnosed by means of cervical cytology as negative to cancer and secondarily the presence of CaCu or other malignant processes and related to cell proliferation was demonstrated by means of histopathological study30, the present invention describes a method of detection of CaCu in early stages by detecting the Eagl protein in cervical tissue, both normal or with diagnosis of premalignant and malignant lesions of the cervix, as well as the detection of said protein in samples of patients exposed to risk factors for the development of CaCu, such as HPV infection and estrogen consumption, with a view to its application in the timely detection of CaCu, improving the sensitivity of traditional cervical cytology without the need to perform additional invasive procedures to patients and thereby reducing the risk of developing invasive carcinoma.

For purposes of the present invention, the expression of the Eagl protein in cervical cancer samples, its presence in cervical samples with alterations in surrounding tissues and the regulation of its gene and protein expression by oncogenes of HPV and estrogens, allow us to think that Eagl fulfills the characteristics of being a marker and indicator of CaCu directly related to the early development of the disease, and without its detection present the problems of interpretation (high proportion of false positives and negatives) associated with diagnostic and detection procedures of CaCu developed so far, so that the detection of Eagl by the method of the present invention, allows to provide a new method of timely detection of CaCu.

The present invention provides a method to detect CaCu from early stages of its development through the detection of the Eagl protein in cells from cervical uterine tissue, because said protein is differentially expressed in smears of normal cervix and in lesions precancerous of the cervix.

In one embodiment of the present invention, the method described herein suggests an association between the expression of the Eagl protein and the development of cervical dysplasia from early stages. The expression of Eagl protein in approximately 10% of morphologically normal cells from intraepithelial lesions could be a very important indicator of the presence of possible lesions in cases in which abnormal cells are not observed in cervical cytology .

As a result of the present invention, an early CaCu detection kit based on the detection of the Eagl protein is proposed. In addition to the patient sample, said detection kit includes adequate positive and negative controls to adequately validate in the clinic the detection of the Eagl protein, using normal CaCu cell lines and keratinocytes, respectively.

Likewise, the method of the invention allows detecting the intracellular localization of the Eagl protein, which is located differentially in the cell in relation to the degree of development of CaCu. In this sense, one embodiment of the method of the present invention is to determine the cellular localization of the Eagl protein, for example, whether in the nucleus or nuclear membrane indicating early stages of the disease (eg, LIE-BG lesions) or in addition to the nucleus, in the nuclear and plasma membranes as well as in the cytoplasm indicating more advanced stages (eg LIE-AG lesions).

In another embodiment of the invention, the method described here allows determining the detection of the Eagl protein in patients who consume estrogens as a possible risk indicator of the subsequent development of cervical uterine lesions.

The present invention also relates to the utility of the detection of the Eagl protein as an early biomarker of CaCu in premalignant lesions of the cervix, which consequently makes it possible to propose a therapy with antibodies directed against this protein providing treatment for CaCu.

In one of the embodiments of the invention, the method described here increases the sensitivity of other tests in the detection of CaCu from early stages, since when combining for example the detection of the Eagl protein in cervical tissue according to the present invention with the HPV detection in the same samples increases the capacity of both tests in the early detection of CaCu. However, the sensitivity values of the detection method of the invention allow CaCu itself to be detected from early stages of its development without having to be combined with other detection methods.

For purposes of the invention, the detection method described herein comprises the steps of: a) Obtain a sample of cervical tissue from a patient and process said sample so that it has the adequate conditions to be analyzed later.

At this stage any technique known in the art can be used to obtain the patient's sample, such as uterine scraping. On the other hand, particularly at this stage, it is preferred to quickly place the brush with which the cells were obtained inside the tube containing the special liquid to preserve the sample under the appropriate conditions (liquid cytology tube). b) Add to the treated sample an antibody that specifically recognizes the Eagl protein and allow said antibody to bind to its target site. In this case it is preferred to add to the sample an antibody capable of specifically binding to at least one epitope comprised in the pore region of the mammalian Eagl protein (amino acids 374-452) and not interacting with the mammalian Eag2 protein.

This step is part of a routine technique in the pathology laboratories in which the incubation of the treated sample with precise dilutions of the antibody is included, which allows adapting the method of the invention to the routine methods of the laboratories of faster. c) Add detection means that reveal the binding of the antibody to its target site.

In this stage it is possible to use different indicators as detection means, which allow to develop a particular signal (for example, coloration or light of a certain wavelength) when the molecule of interest joins its target site, which is processed by standard techniques known in the art, and d) Detect the presence of the Eagl protein in the sample.

In this step, the detection of the signal produced in the previous stage indicates the presence of the Eagl protein in the sample, which can be done visually or by means of detection systems of said signals known in the art.

According to the present invention, the presence of the Eagl protein in the sample analyzed will indicate the presence of cervical intraepithelial lesion associated with the development of CaCu in the patient from early stages; Likewise, the intracellular localization of the Eagl protein in the sample could indicate the degree of development of intraepithelial lesion associated with CaCu in the patient, that is, if the lesion associated with CaCu is in early or early stages, or in advanced stages. This will allow providing follow-up, monitoring and adequate treatment to the diagnosed patient, substantially reducing the risk of developing CaCu in invasive or malignant stages.

As described by Farías et al, 30 it was possible to detect Eagl mRNA in four cervical biopsies obtained by hysterectomy of patients with normal Pap smears (negative to cervical alterations), these samples being from 3 patients (with HPV infection, with tumor). in ovary and with endometrial hyperplasia), which indicated that Eagl could be an early indicator of cervical alterations; However, this study did not include samples of pre-malignant lesions (high or low grade lesions), so this study was not conclusive about the possible use of Eagl as an early marker of cervical alterations.

Unlike that described by Farías et al. 30, the present invention allows detecting the expression of the Eagl protein for the first time in pre-cancerous lesions and directly in exfoliated cells of the cervix obtained by non-invasive routine methods, which makes it possible to propose a kit of diagnosis that can be easily implemented in any laboratory / clinic that performs the Papanicolaou technique. If the detection of the protein was done looking for the presence of Eagl mRNA, the technique would be much more complicated due to the great lability that this molecule presents; likewise, the training and standardization of the technique in common laboratories would require a greater investment of time and infrastructure.

On the other hand, the method of the present invention is focused on clearly identified cervical dysplasia and considers a larger sample size for validation purposes, using cervical tissue samples from patients with high and low grade intraepithelial lesions. Thanks to the method of the invention, it is now possible to associate the expression of Eagl with a well-identified pre-cancerous process and with a sample size that allows us to reduce the individual differences that may arise between patients. The method of the present invention can be carried out through immunoassays, such as for example immunohistochemistry (IHC), immunocytochemistry (ICQ), ELISA (enzyme-linked immunosorbent assay) or any other immunoassay that allows detecting the binding of specific antibodies against the protein Eagl in the samples under analysis, as well as through diagnostic kits (kits) to perform the detection assay of the Eagl protein, for example in situ.

In one of the embodiments of the invention, the immunoassay is performed with a clinical sample of cervical tissue from a patient to stain a portion of the cells from said clinical sample using a specific antibody capable of binding to the Eagl protein. According to the invention, the performance of the immunological assay described here provides such evidence that it allows to relate the positive staining that the clinical samples may present with the progression of the stage of the disease, including a state of early dysplasia (LSIL), or an injury High-grade squamous intracervical (HSIL).

For example, a method for detecting the Eagl protein in cervical tissue samples from a patient includes conducting an IHC assay on a support containing a thin section of the cervical tissue to detect in situ the Eagl protein that may be present in the biological sample. on the support and using an antibody specific for the Eagl protein together with means for detecting the binding of the antibody to the Eagl protein to stain the analyzed section of the tissue.

The binding of the antibody with the Eagl protein present in the biological sample is examined, for example under the microscope or any other detection system that allows to visualize the presence of an agent that reacts with the antibody bound to the protein. In this sense, the agent can be a second antibody or substance that recognizes the first antibody that binds to the Eagl protein, where said agent is conjugated with marker substances, which develop color or light in the presence of colorimetric substances, fluorescent chromogens or substances that decompose by the action of the marking substance emit light of a certain wavelength, or combinations thereof.

As a marker substance of the second antibody for example, it is preferred to use marker substances known in the art that generate color development; Examples of such substances useful for the present invention include horseradish peroxidase (HRP), streptavidin, avidin, alkaline phosphatase, colloidal gold, fluorescent compounds such such as, fluorescein, fluorescent poly-L-lysine sothiocyanate (FITC), rhodamine rhodamine or isothiocyanate B (RITC), biotin and dyes. In the present invention for example, a second antibody conjugated with peroxidase or biotin may be employed.

In the case of the chromogens that can be used in the present invention, these vary depending on the marking substance that generates the color development; examples thereof include S ^ '^^' - tetramethyl benzidine (TMB), 2 / 2'-azino-bis (3-ethylbenzthiazoline-6-sulfonic acid) (ABTS), o-phenylenediamine (OPD) or diaminobenzidine. Preferably, the chromogen is provided in a state in which it is dissolved in a buffer solution (eg, 0.1 M NaAc, pH 5.5). For example, the chromogen TMB is decomposed by peroxidase when it is used as the marker substance conjugated with the second antibody to produce a precipitate that develops color. The level of precipitation of the precipitate that develops color, is observed with the naked eye, in such a way that determines the presence of the Eagl protein.

The washing solution includes a buffer solution of phosphate, NaCl, and Tween 20, although other buffer solutions that allow the elimination of interfering substances from the sample to be analyzed can be used. After binding of the antibody with the Eagl protein, an appropriate amount of the wash solution is added to the reaction mixture where the second antibody will be subsequently joined to the Eagl-first antibody protein conjugate. Washing can be carried out several times, for example 3 to 6 times or those necessary to eliminate interfering substances with detection. Phosphate buffer containing, for example, bovine serum albumin (BSA) can be used as a blocking solution, while a solution of N-sulfuric acid can be used as a stop solution for the enzymatic reaction or color development. For purposes of the invention, the biological sample to be analyzed comprises cervical tissue, cervical cells, cervical tumors, cell cultures, biopsies, and combinations thereof. Likewise, the biological sample can be obtained from a group of people as a general population for a routine exploration of CaCu.

The test in cells of the human subject may comprise a cell from a sample of mucosal cells, such as cervical cells, as well as other tissues such as • from the penis, vulva, anus, rectum and other tissues where the Eagl protein may be detected. All of these samples can be used as a sample in the method of the present invention. Preferably a sample of cells from a patient comprises cervical cells as test cells. Cervical cells can, for example, be present as a histological or cytological specimen. The cytological samples comprise conventional cervical scrapings as well as preparations on thin surfaces of cervical and cervico-vaginal samples or vaginal samples collected by the patient himself.

In another embodiment of the invention, a method for detecting the development of CaCu in a human subject by detecting the Eagl protein in cervical tissue, includes obtaining a clinical sample of the human subject, and conducting one or more immunological assays on the clinical sample. of said subject using specific antibodies against the Eagl protein to detect the development of CaCu from the presence of the Eagl protein in the samples of the subject. Likewise, the Eagl protein in the sample of the human subject can be detected using recombinant antibodies, for example from the Eagl protein described by pardo40,41,42,43,44,45, be polyclonal and / or monoclonal antibodies.

Immunological assays involving antigens, include coating a surface with a primary antibody, such that said antibody captures by affinity and binding the antigen of interest, where said surfaces may be titration plates, membranes, chips, etc. The antigen of interest may be, for example, the complete Eagl protein or unique parts of the protein that allow its specific identification. After blocking the unattached parts of the surface, the clinical sample to be analyzed can be applied for its binding to the antigen capture antibody to form immunocomplexes, which can be detected by another primary antibody or by a detection antibody. join the antigen of interest. Therefore, the two primary antibodies or the pair of both capture and detection antibodies interact with the antigen of interest (sandwich). The capture antibody may be the same or an antibody different from the detection antibody, as long as both antibodies can specifically bind to the antigen of interest.

Subsequently the antigen-antibody complex formed can be detected by a second antibody which has affinity for the detection antibody facilitating the measurement by standard detection systems of immunological complexes using colorimetry, chemiluminescence, fluorescence or various types of substrates. The final readings or visualizations can be done by instruments with appropriate absorbance readers of light or directly by observation and compare the results with the control sample. Positive results will indicate the binding of the antigen of interest to the primary antibody, the capture antibody and the detection antibody and therefore the presence of the antigen of interest in the clinical sample, for example the Eagl protein. On the contrary, negative results will indicate the lack of binding of the antigen of interest to the primary antibodies and therefore the absence of the antigen of interest in the clinical sample, for example the Eagl protein.

One or more immunological assays can be used to detect at least three types of proteins of interest, including but not limited to, antigen, antibody and antigen / antibody immunocomplexes, among others.

The formats of one or more immunological assays can be in microplate format, rapid test based on membranes, protein chip, etc. The principles of the tests are the same that have been described except for the detection systems that vary depending on the chosen substrate to analyze the results in different ways by a specific instrument designed for the test. In summary, the procedures, conditions, binding specificity, development of a type of immunological assay in a format can be adapted in a different format of the same or different immunological assay and / or different immunological assay of the same or different form.

One or more immunological assays according to the present invention lend themselves to using polyclonal and / or monoclonal antibodies, resulting in immunological assays with high sensitivity and specificity to detect the presence of the Eagl protein. Such assays include, but are not limited to, protein chip assays, antigen assays for Eagl, antibody assays for antibodies directed to Eagl, ELISA assays, immunocomplex assays, radioimmunoprecipitation assays, rapid membrane immunochromatographic assays, -IHQ of tissues and / or cervical cells among others and ICQ tests followed by flow cytometry. Likewise, such assays can be non-invasive with minimal or not required additional instruments.

A cell or tissue sample provided preferably as scrapings or other cytological samples obtained from a mammal, preferably a human, is conveniently pretreated to allow contact between a target cell component of a test cell comprised in said sample with an agent that detects Eagl in comparison with that sample of a normal cell that can be compared. Samples can be mounted on a convenient support to allow observation of the cells. Examples of well-known supports include glass, polystyrene, polyethylene, polypropylene, polycarbonate, polyurethane, optionally provided with surfaces to improve cell adhesion and immobilize the sample. Cervical scrapings or biopsies may be prepared as in the Papanicolaou test or by any modification known to any person skilled in the art and may be fixed by procedures that allow appropriate access to the agent or target component. In certain embodiments of the invention, cytological specimens are provided as samples of conventional scrapings or as thin preparations of cervical or liquid cells based on cytological samples or any other preparation known to persons skilled in the art. If storage is necessary, routine procedures using buffers with formalin followed by paraffin coating can be used, which allows the tissue to be well preserved for further analysis. To allow dyeing in the IHQ or In immunofluorescence, the antigenicity of the sample must be recovered and not masked. A method to recover the antigenicity. of the sample preserved by formaldehyde includes the treatment of the sample with proteolytic enzymes, which partially digest the material of the sample by making fragments of the original proteins accessible to the antibodies, or by heat treatment.

The antibodies then come into contact with the sample material in a suitable buffer allowing its binding to the Eagl protein. Until the specific binding of the antibody occurs with the Eagl protein, the antibodies can be detected by methods such as confocal scanning microscopy, brightfield microscopy, flow cytometry or modifications of such techniques, which are known in the art.

For purposes of the present invention, the term "antibody" includes intact antibody molecules as well as fragments thereof, such as Fab, F (ab) 2 or CDR fragments which are capable of binding to the Eagl protein. The antibody as used herein may also refer to another protein or non-protein molecules with an Eagl-binding specificity, such as miniantibodies, aptamers, etc.

Monoclonal antibodies, for example, can be used in the diagnostic method of the present invention, for example in immunoassays in which they can be used in liquid phase or linked to a solid phase carrier. Additionally, the monoclonal antibodies in these immunoassays can be detected by being labeled in several ways. Examples of the type of immunoassays that can use monoclonal antibodies are competitive or non-competitive immunoassays either directly or indirectly. Examples are radioimmunoassays (RIA) and sandwich immunometric assay. The detection of antigens using monoclonal antibodies can be performed using immunoassays that can be performed simultaneously, including IHQ or ICQ of physiological samples. Those skilled in the art will know or be able to discern, that other immunoassay formats can be used.

Monoclonal antibodies can bind to different carriers or be used to detect the presence of Eagl. Examples of known carriers include glass, polystyrene, polypropylene, polyethylene, dextran, nylon, amylases, natural and modified celluloses, polyacrylamides, agaroses and magnetite. The nature of the carrier can be soluble or insoluble for the purposes of the invention. Those with knowledge in the art will know of other suitable carriers to bind monoclonal antibodies.

In carrying out the assays it is desirable to include certain blockers in the incubation medium, usually added with the soluble labeled antibody. The blockers are added to ensure that the non-specific proteins, proteases, or artifact substances present in the experimental sample under analysis do not bind with the antibodies or labeled indicator antibody, which may generate false positive or false negative results. The selection of the blockers can therefore add substantial specificity to the assays described in the present invention. A number of non-relevant (non-specific) antibodies of the same class or subclass of those used in the assays can be used as blockers. The concentration of such blockers (usually from 1 to 100 μg / μl) may be important to maintain the appropriate sensitivity and even inhibit any unwanted interference that may be caused by the proteins contained in the sample.

The specific antibodies against the Eagl protein that can be used in the method of the present invention are for example those antibodies or fragments thereof that specifically recognize the Eagl protein, said protein being a K + ion channel. In this regard, methods for producing specific antibodies against proteins of interest, for example against the Eagl protein, 40,46 are well known in the art, so that it is possible to produce such antibodies.

Examples of antibodies useful for the present invention are those described by Pardo40'41'42'43'44,45 ', which show specificity against an Eagl recombinant protein. However, it is preferred to use the antibodies described by Stühmer46,47 and / or Hemmerlein31, or those used by Gómez-Varela38 and / or Farías30, which are antibodies specific for the Eagl protein (humanized or not) or fragments thereof, such as as CDR fragments, which interact with at least one epitope of the extracellular domain (for example, that comprises the amino acid sequence 374-452 of mammalian Eagl) and / or with an epitope of the intracellular domain (eg, comprising the amino acid sequence 872-932 of mammalian Eagl) of the mammalian ion channel Eagl in a specific manner , and that do not interact with the mammalian ion channel Eag2. In this sense, those antibodies which interact with at least one epitope of the extracellular domain or also called pore region ("pore region of Eagl") comprising the amino acid sequence 374-452 of mammalian Eagl are preferred, for example those described by Hemmerlein31 or the "A" type antibodies described by Stühmer46,47 and which do not bind and / or interact with the mammalian Eag2 protein.

The present invention also provides a diagnostic kit (kit) for performing the detection method of the invention, for example through IHC, to detect the protein. The kit may include a blocking solution prior to the binding of the antibody to the Eagl protein, a blocking solution subsequent to the binding of the antibody to the Eagl protein, a specific anti-Eagl antibody as the first antibody, a second antibody, for example anti- mouse or anti-rabbit conjugated with peroxidase (HRP) or biotin or other agents that reveal the binding of the second antibody with the first antibody, as well as a solution containing appropriate agents used as substrates to detect the binding of the second antibody. The anti-Eagl antibody can be, for example, an antibody specific for the Eagl protein, the antibody described by Sthumer46,47, those used by Gómez-Varela38 and / or Farías30 or a combination of said antibodies. Such a kit can be used to perform an immunological assay, including but not limited to ELISA (enzyme linked immunoabsorbant assays), antigen assays for the Eagl protein, antibody assays for antibodies to the Eagl protein, assays for Eagl immunocomplexes, protein chip assays, radioimmunoprecipitation assays, immunochromatographic assays, tissue IHC and / or cervical cells and immunocytological assays followed by flow cytometry, as well as combinations of such assays.

In this case, the kit may additionally contain tissue samples that can be used as positive or negative controls to validate the results obtained from the detection test of the present invention, such as healthy hippocampal or human placenta tissues or lines HeLa or SiHa cervical cancer cell phones (both commercially available) as positive controls, whereas a normal keratinocyte cell line (commercially available) can be used as a negative control since it does not express the Eagl protein.

The detection of Eagl protein by immunological assays can be used in early clinical inspections to detect CaCu and in general diagnosis for CaCu and can be done by a simple rapid test or by more complete tests that involve the combination of several immunoassays, for example by the simultaneous or parallel detection of the Eagl protein and of HPV in the same samples under test, which significantly increases the sensitivity and accuracy of the detection test, as demonstrated in the present invention (see example 3).

Early diagnosis of CaCu is important for its successful prevention and treatment, which is one of the forms of death from cancer. For example, it is known that importantly an infection is necessary in women aged 12 to 15 years with HPV before developing invasive cancer, therefore it is important to detect early biomarkers to detect the early development of CaCu, which will prevent and treat this cancer early, avoiding as much as possible treatment with chemotherapy or radiation to treat malignant cancer. The development of the immunological assays described here to detect the presence of Eagl as an early biomarker of CaCu development can be used in the population for the early diagnosis of the disease and therefore prevent CaCu. v For the purpose of illustrating the present invention, the following examples are presented, without this implying a limitation to the scope of the invention.

Example 1. Materials and methods. a) Detection of Eagl expression in cervical cytology.

- Obtaining the samples: Cervical cytology: Cytologies were obtained in liquid base by cervical smear of patients who attended the departments of cytology and dysplasias of the General Hospital "Manuel Gea González" of the Secretary of Health, in Mexico City, following the corresponding ethical standards and the following criteria: Inclusion criteria: a) All the patients that come to the opportune detection of the CaCu to the department of cytology, b) All patients who come to the dysplasia service for the first time.

Criteria for non-inclusion: a) Patients who received treatment for cervical dysplasia or for CaCu. b) Patients who did not accept to participate in the study.

Exclusion criteria: a) Samples with insufficient material for morphological analysis. b) Samples that, when processed by ICQ, were observed with the precipitated antibody.

- Processing of the samples: Cervical cytology: The samples were placed in a container with 20 ml of fixation solution (Bio SB, Sta. Barbara CA) and transported at room temperature to be processed.

To fix the cells, the samples were vortexed for 50 sec, the cervical brush was removed and 10 ml of the contents were added in a Falcon tube to which 1.5 ml of density gradient fluid was previously placed (Bio SB, Sta Barbara CA), centrifuged for 10 min at 1,000 g at 25 ° C, the supernatant and the button were removed The cell was resuspended in 1 ml of bidistilled water for each mm of cell concentrate and vortexed for 50 sec. 1 ml of the sample was allowed to settle for 15 min on loaded lamellae (Biocare medical, Concord CA), to subsequently discard the remaining liquid, fix with 96% ethanol and allow to dry in the open air. The lamellae were stored under refrigeration until further processing by means of ICQ with anti-Eagl antibodies to detect Eagl, pl6 (Abcam, Cambridge MA) and HPV (Dako, Carpinteria CA). In this case, the anti-Eagl antibodies used specifically recognize at least one epitope of the pore region of mammalian Eagl (amino acids 374-452) and do not interact with the mammalian Eag2 protein, obtained as described by Hemmerlein31.

- Groups under analysis.

The samples obtained were grouped by cytological diagnosis, according to the Bethesda classification and the consumption or not of estrogen, following the flow diagram shown in figure 1.

- ICQ e lHQ.

In the case of biopsies in paraffin blocks, the slices placed in the lamellae were dewaxed and rehydrated by immersing for 4 to 5 min in successive baths of Xylol, xylol / absolute alcohol (1: 1), absolute alcohol (JT Baker), ethanol (Sigma) and wash buffer (PBS / Twin 20) (Ventana medical, Tucson AZ); In the case of cytologies and cell cultures, they were rehydrated by immersing them from absolute alcohol in a similar way to biopsies.

Subsequently, heat-induced antigenic recovery was performed, placing the lamellae in a solution of 10 mM EDTA, pH 8 in a pressure cooker at maximum temperature, for 30 min., Allowing them to cool in the open air and then placing them in buffer. washed. 100 ml of dual blocker of endogenous peroxidase and phosphatase activity (Dako, cat S2003) was added to the lamellae and incubated for 30 min; Afterwards they were rinsed with washing buffer and 100 ml of the antibody was added primary anti-Eagl (1: 100) mentioned above, for HPV (1:50) or for pl6 (1:50) and allowed to incubate for 60 min.

The samples were subsequently rinsed with washing buffer and 100 ml of biotinylated secondary antibody (mixture of biotinylated anti-mouse / anti-rabbit antibody, IgG sotype) was added.; Ventana medical, Tucson AZ), incubated for 15 min, rinsed and then added 100 ml of streptavidin (Ventana medical, Tucson AZ) and incubated for 15 min. The samples were rinsed with washing buffer, 100 ml of diaminobenzidine was added with 100 ml of H202 (Ventana medical, Tucson AZ) and incubated for 4 min. Finally, the samples were rinsed with washing buffer and 100 ml of copper sulfate (Ventana medical, Tucson AZ) was added and incubated for 5 min.

Tissues and cells treated previously were compared by adding 100 ml of hematoxylin (Ventana medical, Tucson AZ) for 5 min., Rinsing with washing buffer and adding 100 ml of lithium carbonate to turn the color of hematoxylin to blue ( Medical window, Tucson AZ).

The cells and tissues were dehydrated by immersing consecutively in alcohol, absolute alcohol, xylol / absolute alcohol and xylol; they were mounted with the permanent resin for 107960 Entellan microscopy (Merck, Darmstadt) and visualized with a light microscope for evaluation. The sample was considered positive when there was a brown coloration (peroxidase) in the nucleus, cytoplasm and / or cell membrane. b) Statistical analysis.

To calculate the size of the sample, it was decided to apply the central limit theorem48, assuming that a sample of a population behaves similarly to a population with a normal distribution if "n" is greater than or equal to 30, therefore, 30 to 40 samples from each group were processed.

The results obtained were analyzed using the Chi2 (C2) test of Pearspn, using the following formula to evaluate if there was dependence among qualitative or categorical variables.

X2 = S [(Of - E¡) 2 / E¡] where S is the summation, 0j is the observed frequency and Ej is the expected frequency.

Additionally, the risk of the variables studied was studied to increase the expression of the Eagl channels, using the Moment Ratio Analysis (OR), using the statistical program SPSS 15.0.

Validity is the degree to which a test measures what it is supposed to measure, that is, it tells us how often the test result is confirmed by more rigorous diagnostic procedures, the sensitivity and specificity of a test are measures of its validity. The safety of a diagnostic test is determined by the positive and negative predictive values, which tell us with what safety a test will predict the presence or absence of disease and before a positive result of a test tells us the probability that exists that this result indicates presence of the disease49.

Sensitivity is the ability of the test to detect the disease, while specificity is the ability of the test to detect healthy subjects. The positive predictive value is the probability of suffering from the disease if a positive result is obtained in the test and the negative predictive value is the probability that a subject with a negative result in the test is really healthy49.

To calculate the sensitivity (S), specificity (E), positive predictive value (PPV) and negative predictive value (NPV), the following formulas were used50: S = VP / VP + FN E = VN / VN + FP VPP = VP / VP + FP VPN = VN / VN + FN where VP is the number of true positives, FN is the number of false negatives, VN is the number of true negatives and FP is the number of false positives.

Example 2. Detection of Eagl protein expression as a marker of cervical intraepithelial lesions.

As a positive control of Eagl expression, paraffin-fixed hippocampus and placental biopsies were used (Figure 2A and 2B), both tissues previously reported as positive for IHC expression for Eagl31'39. A primary culture of a patient with cervical cancer was also examined (Figure 2C) that was previously reported as positive for Eagl by means of ICQ. Finally, a cervical biopsy with positive LIE-BG for HPV was evaluated (Figure 2D), which showed positivity in the basal and intermediate cell layers.

As negative controls for the ICQ reaction, placental cleavage (Figure 2E) and CaCu primary culture cells (Figure 2F) were used in which antibody diluent was applied in place of the primary antibody to discard any specific label by the reagents used during the IHQ process.

Because an increase in mRNA expression has been reported in keratinocytes expressing HPV oncogenes or one of their oncoproteins, the expression of Eagl channels at the protein level was explored in normal keratinocytes (Figure 3A) and in human keratinocyte cell lines transfected with the E6 oncoproteins (Figure 3B) and E6 / E7 (Figure 3C) of HPV16, as well as in HPV16 positive CaCu cell lines (HeLa) (Figure 3D) and HPV18 (SiHa) (Figure 3E) 39'51. As can be seen in Figure 3, normal keratinocytes do not express the Eagl protein, while the rest of the cell lines studied were positive to Eagl, presenting a distribution mainly at the nuclear level. These observations were extremely important, since they allowed us to conclude that normal keratinocytes and the HeLa and SiHa tumor lines can serve as excellent negative and positive controls, respectively, as part of a diagnostic kit.

Additionally, in order to further confirm the specificity of the antibody used to detect Eagl potassium channels, ICQ was performed. on the cell line of CaCu positive for HPV18 (SiHa) as well as for keratinocytes E6 and E6 / E7 in the presence of the antibody alone and with a specific fusion protein that contains an area close to the putative region of the pore and that has been used previously as a control of the specificity of said antibody38, for the epitope detected by the antibody, finding that in both cases the mark produced is completely blocked in the presence of only the antibody (Figure 3F).

To evaluate the expression of Eagl in premalignant lesions of the cervix, a total of 151 samples were collected, of which 93 were from patients with a cytological diagnosis of normal cervix, 2 from ASC-US (atypical squamous cells of undetermined significance) and 65 from patients diagnosed with cervical dysplasia; of the normal samples 10 were reclassified to low-grade dysplasia after HPV evaluation. The 2 ASC-US, 16 dysplasias and 3 normal samples were eliminated because they contained insufficient material for the cytological study or for precipitation of the antibody. The average age of the patients with a normal cervix diagnosis was 41 years, with an LIE-BG was 35 years, and with a LIE-AG of 40 years.

ICQ was performed for Eagl in a total of 130 cervical cytology samples, of which 81 samples corresponded to a cytological diagnosis without evidence of cervical (normal) injury and 49 samples to cervical intraepithelial lesions (dysplasias), of which 36 samples were LIE-BG and 13 LIE-AG samples (Table 2), which found that 71% of the samples from the normal cervix were negative to Eagl (Figure 4A) and 29% of them expressed the Eagl ion channel (Figure 4B) with a predominant expression in the cytoplasm and in the nuclear membrane. The cervical intraepithelial lesions (dysplasias) were positive in 73% of the samples, expressing mainly in the cytoplasm and in the nuclear membrane in the low-grade lesions (Figure 4C) (67% positive), while the high-grade lesions (92% positive) showed a more widespread subcellular distribution, including nucleus, cellular and / or nuclear membranes and cytoplasm (Figure 4D).

In addition, it was observed that in samples from patients with diagnoses of both low and high grade IEL, cells that morphologically considered normal during the Papanicolaou study, were positive for the antibody for Eagl (Figure 5). This observation is of additional importance because if in a cytology only morphologically normal cells are collected despite the existence of an intraepithelial lesion, said sample is commonly reported as normal. The expression of Eagl in some morphologically normal cells may be an element to indicate a closer monitoring of the patient.

Table 2 ICQ in cervical cytology * Performed by Papanicolaou.

NR Unrealized.

Example 3. Detection of Eagl protein expression in cervical cells and association with HPV infection.

Due to the importance of the human papilloma virus in cervical carcinogenesis and to I recent evidence of the expression at the level of R Am of Eagl in cell lines d cervix carcinoma positive to HPV39, a comparison was made by means of ICQ of the expression of Eagl, Ll of HPV and pl6 in cytologies with diagnosis of dysplasia (Table 2). It was found that 73% were positive for Eagl, 71% for HPV (Figure 6A) and 48% were positive for p6 (Figure 6B). As can be observed, the detection of Eagl was greater than the detection of HPV or of p6, the latter 2 being currently used in the clinic for the diagnosis of cervical lesions and CaCu. It was also observed that the detection of HPV by ICQ was lower than expected, since the reported incidence of HPV infection in cervical cytology is almost 99% for LIE-AG and 80% for LIE-BG. As positive controls for the anti-HPV antibody, a cut of a paraffin-embedded HPV infection biopsy was used (Figure 6C) and HeLa cells were used as control of the ICQ for p6 (Figure 6D).

Additionally, the probable association between the presence of HPV infection and Eagl expression was evaluated, with Eagl expression observed in 69% of HPV positive samples, while only 23% of HPV negative samples were positive for Eagl, which showed an association of Eagl expression with HPV infection. Knowing that the detection of HPV by molecular techniques increases the sensitivity of the detection of cervical intraepithelial lesions52, all the patients that were positive for HPV were grouped by PCR together with those with a diagnosis of cervical intraepithelial lesion by cervical-vaginal cytology. which was found to have a greater association of the presence of cervical disease (intraepithelial lesion and / or HPV infection (HPV)) with a greater expression of Eagl, finding that 71% of the samples with disease are positive to Eagl, in against 15% in samples without cervical disease. The found values of X2 and the odds ratio (OR) (table 3) strongly suggest the association between the parameters studied (Eagl, HPV and disease).

Table 3 Although it is necessary to perform a more complete validation that includes biopsies of high and low grade intraepithelial lesions and an even more detailed detection of HPV types, the data shown here propose the Eagl channel as a possible marker of cervical intraepithelial lesions. . When combining both methods (HPV detection and otological study) 50, preliminary validation data were found that are adequate to use the antibody for Eagl as an auxiliary tool in the timely detection of CaCu and its early lesions (Table 4).

Table 4 The detection of Eagl by means of the method of the present invention in biopsies of the cervix with diagnosis of intraepithelial lesion, showed positivity in the basal and intermediate layers of the cervical epithelium, which correlates adequately with the results obtained in cervical cytologies, since Eagl expression was found more frequently in the intermediate cells. In the case of cytologies performed conventional way for the diagnosis of CaCu, it is impossible to show the cells coming from the basal layer since these cells do not detach, so the method of the present invention allows to explore this type of cells increasing the sensitivity of the test.

In accordance with the high expression of the Eagl channels reported at the mRNA level of cell cultures of CaCu positive for HPV and in keratinocytes expressing the HPV oncoproteins1632'39'51, the detection of the Eagl protein in cervical cells by the method of the invention was positive in all cases, showing positivity at the nucleus level as in the primary cultures of CaCu. The presence of the Eagl protein in these cells strongly suggests that the HPV E6 and E7 oncoproteins increase or induce the expression of Eagl, since, on the contrary, normal keratinocytes do not show expression of the Eagl protein.

According to the method of the present invention, the expression of the Eagl protein in cervical cells is associated with the presence of cervical intraepithelial lesion, as well as with the degree of cervical lesion, finding a greater association between high-grade dysplasias than with those of low grade; however, it does not allow us to discriminate adequately between the different degrees of injury, since the Xi2 values are not significant between the low and high-grade lesions.

However, the differential localization in the cervical tissue of the detected expression of the Eagl protein by the method of the invention can make it possible to detect the different stages of the cervical lesion, since in the initial stages of the disease (LIE-BG) it was observed that the expression of the Eagl protein is found primarily in the nucleus and the plasma membrane, while in more advanced stages (LIE-AG) the expression of the Eagl protein extends to other locations, including in addition to the nucleus, the nuclear and plasma as well as the cytoplasm. The expression of Eagl protein in the nucleus in early stages of the disease, could be explained because Eagl could be participating as a transcription factor, stimulating genes that lead to the transformation and proliferation of cervical cells. This intracellular distribution, can be explained because the gene of eagl presents a nuclear signaling sequences that would allow it to translocate to the nucleus and possibly bind to DNA, which could allow some transcription factors to be activated in a similar way to CavL2 channels, whose translocation its carboxyl terminal segment towards the nucleus stimulates transcription53.

One of the main advantages of the method of the present invention is the detection of the Eagl protein in cells with normal morphology that were found in the samples with diagnosis of cervical lesion. In this case, remembering that one of the main problems in the detection of cervical cancer in the high number of false negatives, because during the sample collection may exist mostly unaltered cells, being able to detect the Eagl protein indicates that there is a damage at the molecular level in the cervical tissue, which would allow close monitoring of the patient who provided the sample.

Validation of the use of anti-Eagl protein antibodies in the detection of cervical intraepithelial lesions from early stages due to the values of sensitivity and specificity observed in the method of the invention, in accordance with that reported for other antibodies currently used for the detection of CaCu in the Clinically, it allows the use of specific antibodies for the Eagl protein as a marker of cervical lesions or of a greater risk for its development; additionally, it offers a possible therapeutic tool, since being a target of membrane and with the antecedents that the blocking of its activity decreases the cell proliferation In Vitro as well as of tumors in vivo, the use of antibodies directed against the Eagl protein could contribute to cytoreduction of tumors, for example in CaCu.

Example 4. Detection of the expression of the Eagl protein in cervical cytologies and its association with the consumption of estrogens.

Since long-term estrogen use is considered a possible risk factor for CaCu and estrogen induces the expression of eagl genes in vitro, expression was explored of normal Eagl in this type of cytology of patients who use estrogen, either as part of a hormone replacement therapy or as part of a contraceptive treatment. As can be seen in table 5, the expression of Eagl in these cytologies was found in almost 50% of the samples analyzed; likewise in most cases, immunostaining to Eagl was observed in the nucleus (data not shown). Statistical analysis showed an association between estrogen use and Eagl expression (X2 = 9.68), while calculated OR values strongly suggested estrogen consumption as a factor that significantly affects the expression of Eagl in cervical tissue.

Table 5 Expression of Eagl in normal cervical cytologies of patients using estrogen The results shown here demonstrate that Eagl expression is possibly a risk indicator for the development of cervical uterine lesions in patients who consume estrogen. In this case, a broad study of the population is also necessary to determine the possible association between the expression of Eagl and the duration or type of estrogen used (injected, oral, etc.). Similarly, a prospective study is also necessary to know what percentage of patients who consume estrogen and who shows the expression of Eagl develop cervical uterine lesions. It would also be very interesting to explore the expression of Eagl in other conditions where estrogenic imbalances are found, for example, in some obese women. However, these results suggest Eagl as a possible risk indicator for the development of cervical uterine lesions in patients who consume estrogen.

One of the possible explanations for the presence of Eagl-positive cells in samples from patients diagnosed with normal cervix is the level of physiological estrogen or hormonal consumption exogenously as part of hormone replacement therapy and as contraceptives, It is known that estrogens have been linked to the development of different types of cancer, including CaCu.

Our observations highlight the emerging emergence of Eagl as an early tumor marker30'31'34. In particular, the method of the present invention in the monitoring of CaCu in prevention programs could contribute to improve the timely diagnosis in early stages and to reduce the mortality of this disease.

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Claims (23)

1. A method for detecting cervical cancer from early stages of its development in a human subject, characterized in that it comprises the steps of: a) Obtain a sample of cervical tissue from the human subject, b) Add to the sample an antibody capable of binding to the Eagl protein and allowing said antibody to bind to its target site, c) Add to the sample of step b) detection means that reveal the binding of the antibody to its target site, and d) Detect the presence of the Eagl protein in the sample, where the positive detection of the presence of the Eagl protein is related to the development of cervical uterine cancer in the human subject.
2. The method of claim 1, characterized in that the antibody is capable of specifically binding to at least one epitope comprised in the pore region of the mammalian Eagl protein (amino acids 374-452) and does not interact with the mammalian Eag2 protein.
3. The method of claim 1, characterized in that the antibody is selected from the group comprising polyclonal antibodies, monoclonal antibodies, recombinant antibodies, fragments thereof such as Fab fragments, F (ab) 2 fragments, CDR fragments, and mixtures thereof .
4. The method of claim 1, characterized in that the sample is obtained by scraping the cervical tissue.
5. The method of claim 1, characterized in that the cervical tissue is selected from the group comprising cervical cells, cervical tumors, cell cultures, biopsies, and combinations thereof.
6. The method of claim 1, characterized in that the detection means comprise detection systems that allow to visualize the presence of an agent that reacts with the antibody bound to the Eagl protein.
7. 7. The method of claim 6, characterized in that the agent is selected from the group comprising a second antibody conjugated with a colorimetric agent, a second antibody conjugated with a fluorescent chromogen, a colorimetric agent, a fluorescent chromogen or combinations thereof.
8. 8. The method of claim 1, characterized in that it is carried out through an immunological assay selected from the group comprising ELISA, radioimmunoassay, immunocytochemistry, immunohistochemistry, radioimmunoprecipitation, cytometry. flow and combinations thereof.
9. 9. The method of claim 8, characterized in that the immunological assay is immunohistochemistry.
10. 10. The method of claim 1, characterized in that the immunological assay is performed on a clinical sample of cervical tissue of the human subject and a portion of the cells of said tissue is stained using at least one antibody capable of specifically binding to the Eagl protein and means that reveal the binding of the antibody to its target site.
11. 11. The method of claim 10, characterized in that a positive staining of the nucleus and / or cellular membrane of the cells indicate early stages of the development of cervical uterine cancer.
12. 12. The method of claim 10, characterized in that a positive staining of the nucleus and / or nuclear membrane and / or plasma membrane and / or cytoplasm of the cells indicate advanced stages of the development of cervical cancer.
13. 13. The method of claims 10 to 12, characterized in that the immunological assay is immunohistochemistry.
14. 14. A diagnostic kit for detecting cervical cancer from early stages of its development in a human subject, characterized in that it comprises: a) An antibody capable of specifically binding to the Eagl protein, b) Detection means that reveal the binding of the antibody to its target site, c) Positive and negative controls that allow the validation of the result obtained by the detection means.
15. 15. The diagnostic kit of claim 14, characterized in that the antibody is capable of binding to at least one epitope comprised in the pore region of the mammalian Eagl protein (amino acids 374-452) and does not interact with the mammalian Eag2 protein.
16. 16. The diagnostic kit of claim 14, characterized in that the antibody is selected from the group comprising polyclonal antibodies, monoclonal antibodies, recombinant antibodies, fragments thereof such as Fab fragments, F (ab) 2 fragments, CDR fragments, and mixtures thereof. the same.
17. 17. The diagnostic kit of claim 14, characterized in that the detection means comprise detection systems that allow to visualize the presence of an agent that reacts with the antibody bound to the Eagl protein.
18. 18. The diagnostic kit of claim 17, characterized in that the agent is selected from the group comprising a second antibody conjugated with a colorimetric agent, a second antibody conjugated with a fluorescent chromogen, a colorimetric agent, a fluorescent chromogen or combinations thereof.
19. 19. The diagnostic kit of claim 14, characterized in that the positive controls are selected from the group comprising healthy human hippocampal tissues, healthy human placental tissues, cervical uterine cancer cell lines, cervical cancer cells and mixtures thereof. same.
20. 20. The diagnostic kit of claim 14, characterized in that the negative controls are selected from the group comprising normal keratinocyte cells and mixtures thereof.
21. 21. The diagnostic kit of claim 14, characterized in that it is used through an immunological assay selected from the group comprising ELISA, radioimmunoassay, immunocytochemistry, immunohistochemistry, radioimmunoprecipitation, flow cytometry and combinations thereof.
22. 22. The diagnostic kit of claim 21, characterized in that it is used through immunohistochemistry.
23. 23. The use of the human Eagl protein as an early marker of the development of cervical cancer in a human subject.