Method for improved diagnosis of dysplasias

Description of the invention

The present description relates to a method for better diagnosis of dysplasia, consisting in the simultaneous detection of INK4a gene products and at least one marker of cell proliferation. It is a method that allows to distinguish between dysplastic cells overexpressing INK4a gene products from cells overexpressing INK4a gene products, which are not dysplastic, by detecting a marker suitable to assess the proliferative properties of the cell. Characterization of the proliferative properties may include the detection of a marker or group of markers characteristic of active cell proliferation and / or a marker or group of markers characteristic of delayed or terminated cell proliferation. The method presented here allows for specific diagnostics of dysplasia in histological and cytological samples.

It has been shown that the detection of p16 overexpression^INK4a in biological samples it is a useful marker in the diagnosis of anorectal lesions such as cervical cancer (see WO00 / 01845; Klaes et al., Int. J. Cancer: 92, 276-284 (2001)). The method is based on p16 specific immunochemical staining^INK4a allows for sensitive and specific identification of cells in a tissue section and in cytological samples.

In tissue immuno-histochemistry, dysplastic and neoplastic cells can be stained by staining with specific anti-p16 antibodies^INK4a. Histological diagnosis of neoplastic lesions can thus be confirmed by means of staining based on a molecular marker, characteristic of the transformation of cells in lesions in the anogenital area. In this procedure, the recognition of whether or not cells are cancer cells is not based solely on p16 specific staining.^INK4abut also takes into account histological information.

This is because in approximately 20-30% of the samples, metaplastic cells exhibit some degree of immunoreactivity with anti-p16 antibodies.^INK4a and are stained during the procedure. The staining pattern of metaplastic cells, however, differs from that obtained from neoplastic lesions. Metaplastic cells produce patchy or focal staining, while neoplastic lesions produce a diffuse staining pattern. Moreover, the staining intensity in metaplastic cells is usually lower than in neoplastic cells.

Common methods used in screening tests for the early detection of dysplasia and / or neoplastic lesions do not use histological tests and are usually limited to cytological procedures. However, especially in cases where no histological information on tissue structure is available, such as in cytology, the p16 overexpression alone^INK4a may give false positive results. This is due to the fact that p16 overexpression is the part of metaplastic cells^INK4a at a detectable elevated level, may not be differentiated according to histological criteria.

Percentage of cells overexpressing p16^INK4a increases as dysplasia develops. Therefore, in the neoplastic or pre-neoplastic stages, when there is only a limited population of neoplastic and pre-neoplastic cells in the samples, the immunoreactivity of p16^INK4ashe may be weak. This weak immunoreactivity may be about the same as that of metaplastic cells. In minor stages of dysplasia, total p16 immunoreactivity^INK4a it is stronger and then neoplastic changes can be easily distinguished from metaplasia even in a cytological examination. This can lead to cases where the presence of p16 expressing metaplastic cells is present^INK4a can be considered the presence of cancer cells, leading to false-positive results.

This situation is very unfavorable, especially in screening tests, where it is important to detect the early stages of neoplastic changes. This is particularly important due to the fact that p16-based diagnosis has been shown^INK4a is a useful tool in histological research and that its use in cytology based screening procedures could improve established procedures. To reduce the number of false-positive results in cytological tests and to further increase the accuracy of the diagnosis of lesions in the anogenital area based on p16^INK4a, a differentiation method would be needed to distinguish metaplasia from neoplastic and dysplastic lesions. The method presented here serves this purpose.

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To confirm the distinction between metaplasia and neoplastic changes in procedures based on overexpression of p16^INK4a, a marker molecule, present in neoplastic and pre-neoplastic cells and tissues, which is not present alongside the INK4a gene products in a single metaplastic cell, would be necessary.

The solution to the problem presented here is provided by the methods described here. In the course of the experiments leading to this discovery, researchers showed that simultaneous detection of the presence or absence and / or the level of p16^INK4a and at least one cell proliferation marker such as, e.g., Ki67, Ki-S2, mcm5 or mcm2 can solve this problem.

The article presents several documents on the combined use of molecular markers for better diagnostics of dysplasia. WO0208764 discloses a method for improved diagnosis of cervical malignant lesions using the combined use of an HPV marker and a marker for cell proliferation or viral activity. p16^{! NK4a} is mentioned in the context of this invention as a marker that can be used simultaneously with HPV markers.

EP1217377 shows a method for the automated detection of cervical malignancies by detecting more than one marker molecule. Some defined marker combinations are specified later in this document. However, the document does not address the selection of appropriate markers for combined use. The purpose of the combined use of markers in this application is to improve the accuracy of automated analysis of staining images in biological cytology specimens. The document mentions a p16 connection^INK4a with other tumor markers.

WO02059616 discloses a method for the detection of cell cycle disorders to improve the diagnosis of cervical malignancies. The document describes that dysplastic cells exhibit disturbances in the control of the cell cycle and can therefore be identified by detecting a cyclin E-type protein together with post G1 substances.

Jeffrey Keating in the article "Ki67, Cyklin E, and p16INK4a Are Complimentary Surrogate Biomarkers for human papilloma Wirus-Related Cervical Neoplasia" (American Journal of Surgical Pathology 25 (7): 884-891, 2001) presents the complementarity of the use of p16^INK4a and cyclins in the diagnosis of cervical dysplasia. The document addresses the problems of each single marker used in the detection of dysplasia and states that the use of p16^INK4a together with cyclin E can help to overcome the inconvenience of using single marker particles, especially when evaluating cytological samples. However, no information was given on the use of p16^INK4a together with a marker specific to proliferating cells such as Ki67. The document does not indicate the simultaneous use of p16^INK4a in diagnostic methods; Moreover, it refers to the limited use of Ki67 in the differential diagnosis of cervical lesions and proposes to abandon the use of this marker in the diagnosis of cervical malignancies.

There is no information in this article regarding the combined use of p16^INK4a and a marker characteristic of cell proliferation as a diagnostic method for better differentiation of non-dysplastic cells producing p16^INK4a from dysplasia producing p16^INK4a. WO02059616 gives no guidance on the use of p16^INK4a in detecting proliferation of dysplastic cells. EP1217377 does not disclose the purpose of the combined use of tumor markers for detection other than to automate the analysis process. There is no statement regarding the benefit of the combined use of markers for the purpose of distinguishing between dysplastic cells and e.g. metaplastic cells in cervical-derived samples.

The authors of this method tried to overcome the shortcomings that p16^INK4awhich is overproduced in various dysplasias, may also be detected in some other non-dysplastic cells. The distinction between p16 producing non-dysplastic cells^INK4a and dysplastic cells overproducing p16^INK4a may be based on the proliferation characteristics of the respective cells. In properly controlled p16 cells^INK4a it inhibits cdk4 and thus inhibits proliferation. On the contrary, in dysplastic cells this regulation is impaired. Hence p16^INK4a despite its extremely high expression, it does not inhibit cell proliferation.

The inventors of this method found that dysplastic cells can be distinguished from cells exhibiting controlled cell proliferation by simultaneous detection of p16^INK4a and a marker characteristic of cell proliferation. Due to the fact that in normal cells it ra4

Increased levels of p16^INK4a inhibit proliferation, cells overexpressing p16^INK4a can be classified as dysplastic if they show signs of active cell proliferation.

The invention relates to a method for distinguishing dysplastic cells overexpressing INK4a gene products from other cells expressing the INK4a gene products at a level that is detectable in biological samples, comprising determining the expression of marker particles by cytological or histological procedure, characterized in that simultaneous expression is determined at least two marker particles in at least one single cell, wherein at least one marker molecule is the expression product of an INK4a gene selected from the group consisting of p16^INK4a and p14ARF and the at least one other marker molecule is a cell proliferation marker, and the cell proliferation marker is selected from the group consisting of a proliferation marker necessary to maintain cellular proliferation, a proliferation marker involved in DNA replication, a proliferation marker that is part of a replication fork, or encodes a protein involved in including replication fork, cell aging marker, cell cycle arrest marker and apopotosis marker, wherein overexpression of at least one INK4a gene product selected from the group consisting of p16^INK4a and p14ARF and expression at a detectable level of at least one marker of active cell proliferation within said single cell is indicative of a dysplastic state of the cell, and overexpression of at least one INK4a gene product selected from the group consisting of p16^INK4a and p14ARF and expression at a detectable level of at least one marker of senescence, terminal cell differentiation, apopotosis or cell cycle inhibition, within said single cell, is indicative of a non-dysplastic condition of the cell.

Preferably, a set of two or more cell proliferation markers is detected.

Preferably, at least one INK4a gene product has a molecular weight between 13 and 19 kDa.

Preferably, the product of a gene necessary for maintaining cell proliferation is a molecule selected from the group consisting of Ki67, Ki-S5 and Ki-S2 molecules.

Preferably, the gene product involved in DNA replication is selected from the group consisting of helicases or subunits thereof, cell division cycle (cdc) molecules, phosphatase molecules and kinase molecules.

More preferably, the helicases or their subunits are selected from the group consisting of MCM2, MCM3, MCM4, MCM5, MCM6, MCM7 and HELAD1.

More preferably, cdc, kinase and phosphatase molecules are selected from the group consisting of CDC6, CDC7 protein kinase, Dbf4, CDC14 protein phosphatase, CDC45 and MCM10.

Preferably, the molecules included in the replication forks are selected from the group consisting of PCNA and POLD.

Preferably, the gene product is a polypeptide or nucleic acid molecule.

Preferably, at least one marker molecule is additionally detected in order to improve the assessment of the diagnosis or the prognosis.

More preferably, the additional marker molecule is at least one additional proliferation marker molecule.

More preferably, the additional marker molecule is selected from the group consisting of an aging marker, an apopotosis marker, a cell cycle inhibition marker, a cellular terminal differentiation marker, a viral infection marker, a viral activity marker, a cell cycle regulatory protein, a gene product necessary to maintain cell proliferation, a gene involved in DNA replication, a gene product that is part of the replication forks.

Preferably, a cytological staining procedure using at least one dye from the group consisting of DAPI, Quinacrin, chromomycin, Alan, acridine orange, hematoxylin, eosin, Sudan red, toluidine blue and thionine or a staining method selected from the group consisting of Pap staining, Giemza staining is additionally used. , hematoxylin-eosin staining, van-Gieson staining, Schiff staining, staining with metal precipitates, Tumbulls blue staining and metal cyanide staining.

Preferably, the dysplastic cells are cancerous or precancerous cells.

More preferably, the dysplastic cells are derived from dysplasia associated with papillomavirus infection.

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More preferably, the papillomavirus is a high-risk papillomavirus selected from the group consisting of HPV 16, HPV 18, HPV 31, HPV 33, HPV 35, HPV 39, HPV 45, HPV 51, HPV 52, HPV 56, HPV 58, HPV 59, HPV 66 and HPV 68.

Preferably, the lesion is selected from the group consisting of anorectal lesions, lesions of the respiratory tract, and lesions of the skin and its appendages.

Preferably, the lesion is selected from the group consisting of lesions originating in the cervix, vagina, vulva, penis, anus, rectum, bronchial tree, lung, peritoneal space, nasopharynx, mouth or skin.

Preferably, the biological sample is a sample comprising cells derived from the anogenital area, from the respiratory tract, or from the skin and its appendages.

More preferably, the cells are cells derived from the cervix, vagina, vulva, penis, anus, rectum, bronchial tree, lung, nasopharynx, mouth or skin.

Preferably, the biological sample is a cytological or histological preparation.

Preferably, the detection of the INK4a gene products and / or the cell proliferation marker particles is performed with at least one probe specifically recognizing at least one of the particles to be detected.

More preferably, at least one probe is detectably labeled.

More preferably, the at least one label is a radioisotope, a bioluminescent compound, a chemiluminescent compound, a fluorescent compound, a metal chelate or an enzyme.

Preferably, at least one probe is a protein and / or a nucleic acid.

More preferably, the at least one probe is an antibody directed against the gene product encoded by INK4a or the product of a cell proliferation marker gene.

Preferably, the method comprises an immunocytochemical staining procedure.

Preferably, at least one probe is a nucleic acid that specifically hybridizes to the INK4a gene product or the cell proliferation marker gene product.

Preferably, the method comprises in situ hybridization reactions.

Preferably, the method comprises nucleic acid amplification reactions.

Preferably, the nucleic acid amplification reaction is PCR, NASBA or LCR.

Preferably, detection reactions using nucleic acid probes and polypeptide probes are performed simultaneously.

Another object of the invention is a kit for carrying out the present method, characterized in that it is a diagnostic or research kit comprising at least one probe for detecting the presence or absence and / or level of overexpression of at least one INK4a gene product selected from the group consisting of p16^INK4a and p14ARF, and at least one cell proliferation marker gene product in biological samples, wherein the cell proliferation marker is selected from the group consisting of a proliferation marker necessary to maintain cellular proliferation, a proliferation marker involved in DNA replication, a proliferation marker component of a replication fork, or encoding a protein included in the replication forks, a marker of cellular aging, a marker of cell cycle arrest and a marker of apoptosis.

Preferably, the cell proliferation marker gene products are selected from the group consisting of CDC6, MCM3, MCM4, MCM5, MCM6, MCM7, CDC7 protein kinase, Dbf4, CDC14 protein phosphatase, CDC45 and MCM10, Ki67, Ki-S2, PCNA or POLD.

Preferably, the kit further comprises at least one of the following:

a. sample p16^INK4a to perform a positive control reaction,

b. p14ARF sample to run the positive control reaction

c. Sample Ki67 to run the Positive Control reaction

d. a Ki-S2 sample to perform the positive control reaction

e. MCM5 sample to run the Positive Control reaction

f. MCM2 sample to run the Positive Control Reaction

g. PCNA sample for positive control reaction

h. Reagents for detecting the presence or absence and / or level of p16^INK4a

i. Reagents for detecting the presence or absence and / or level of p14ARF

j. Reagents for detecting the presence or absence and / or the level of Ki67

k. Reagents for detecting the presence or absence and / or level of Ki-S2

I. reagents to detect the presence or absence and / or level of MCM5

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m. Reagents for detecting the presence or absence and / or level of MCM2

n. reagents for detecting the presence or absence and / or level of PCNA

o. One or more samples of the INK4a gene products to run a positive control reaction

p. one or more samples of the cell proliferation marker gene products to perform a positive control reaction

q. one or more reagents for detecting the presence or absence and / or the level of INK4a gene products

r. and one or more reagents for detecting the presence or absence and / or level of other cell proliferation marker gene products.

This method is used to distinguish neoplastic, pre-neoplastic and / or dysplastic lesions from non-dysplastic cells showing high levels of p16^INK4a in biological samples by histological or cytological procedures to detect the presence or absence of cells expressing the p16 gene^INK4a simultaneously with markers of cell proliferation. Suitable markers of cell proliferation are e.g. Ki67, Ki-S2, KiS5, mcm5 or mcm2. In one variation of the method, a protein or mRNA of cell proliferation markers can serve as a marker to distinguish metaplasia from early dysplastic or precancerous lesions in samples.

One aspect of this method is to distinguish dysplastic cells overexpressing INK4a gene products from other cells expressing detectable levels of INK4a gene products in biological samples by determining the simultaneous expression of at least two marker particles in at least one separate cell by cytological or histological procedures. . The at least one marker molecule should be a product encoded by the INK4a gene and at least one other marker molecule should be a marker of cell proliferation. Overexpression of at least one INK4a gene product and expression of at least one marker of active cell proliferation at a level detectable by immunochemistry within a given single cell indicates a dysplastic state of the cell, while overexpression of at least one INK4a gene product and expression of at least one aging marker, final cell differentiation, apoptosis or cell cycle inhibition at a detectable level within a single cell indicates that it is not a dysplastic cell. The second aspect of the present method relates to a test kit for determining dysplasia in samples according to the presented method.

In the course of the experiments leading to the establishment of this method, it was found that in certain situations, non-dysplastic cells may exhibit p16 immunoreactivity.^INK4a. The method founders found that these cells, showing structured control of cell proliferation, were inhibited in growth in response to increased levels of the INK4a gene products. Therefore, these cells do not show immunoreactivity as markers of cell proliferation. In contrast, abnormal cells overexpress p16^INK4a characterized by aberrant regulation of cell proliferation control and not responding to elevated p16 levels^INK4a cessation of proliferation. Thus, dysplastic cells express p16 simultaneously^INK4a and markers of cell proliferation. The researchers found that the simultaneous detection of p16^INK4a and markers of cell proliferation can be used to distinguish dysplastic cells from growth inhibited cells overexpressing p16^INK4asuch as, for example, metaplastic cells.

The discovery that p16^INK4a overexpressed in various dysplasias can also be detected in some other non-dysplastic cells, led the authors of the presented method to establish a technique for distinguishing non-dysplastic cells that produce p16^INK4a from dysplastic cells overexpressing p16^INK4a, based on the proliferation characteristics of the cells in question. Whereas in normally controlled cells, p16^INK4a it inhibits cdk4 and thus inhibits cell proliferation, this is not observed in dysplastic cells. Thus, in p16 dysplastic cells^INK4a despite its extremely high expression level, it does not inhibit cell proliferation.

The method presented here is based on the fact that dysplastic cells can be distinguished from cells showing normally controlled cell proliferation by the simultaneous detection of an INK4a gene product such as p16^INK4a and a marker characteristic of cell proliferation.

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The term marker as well as marker molecule will generally be used herein to refer to the expression products of the proliferation marker gene as well as the expression products of the INK4a gene.

The nomenclature of genes used in this text may in part refer to genes or proteins found in various organisms. In the context of this method, this nomenclature refers to the corresponding homologue of specific markers in the organism for which the method is described. In some embodiments of the present method the organism is a mammal, in one embodiment it may be a human. Thus, in one embodiment of this method, the specified markers will be human homologues of the appropriately defined markers.

In general, throughout the text, the term "(cellular) proliferation marker" or "cell proliferation marker" in various grammatical forms is used to denote proteins and nucleic acid markers. In the case of a protein, a marker name such as, for example, "replication protein" is used, and this usage will be understood as metonymic and will refer to both the protein and the marker particles of the nucleic acid encoding the protein in question.

The marker useful in the present method may be any molecule that is transcribed from the gene, or any molecule that is translated after such transcription. Accordingly, the term gene product as used in the context of the present method may include polynucleotides, such as, e.g., DNA or RNA, and polypeptides, such as proteins, proteoglycans, peptides, etc. The term expression product (s) as used in the context of the present method shall include any transcription product of the gene locus in both directions, including any reading frames, splice variants.

The term expression products as used herein shall include any alternative products encoded by the nucleic acids at the locus of the gene in question.

The term INK4a gene products, as used in the context of the present method, shall be any mRNA transcribed from the INK4a gene locus or any polypeptide translated from such mRNA. In one embodiment of the method, the INK4a gene expression products may have a molecular weight on the order of 5 to 40 kDa, or any value between, preferably on the order of 10 to 20 kDa, or any value between and preferably on the order of 14 to 19 kDa or any value in between.

Suitable INK4a gene products for the present method may include, e.g., gene products such as, e.g., p16^INK4a and p14ARF.

The term "(cellular) proliferation marker" or "cell proliferation marker" as used in the context of the present method will include any marker molecule known to be characteristic of a cell proliferation state. The state of proliferation can be, e.g., the state of active cell proliferation, delayed cell proliferation, inhibited cell proliferation, cell aging, terminal cell differentiation, apoptosis, etc. In one embodiment of the invention, the marker of cell proliferation is a marker molecule characteristic of active cell proliferation. In another embodiment of the method, the proliferation marker molecule may be a molecule that is characteristic of proliferative inhibited, terminally differentiated, senescent, or apoptotic cells.

In some embodiments, the proliferation markers used in the context of the present method may include genes involved in DNA replication, such as, for example, proteins of the pre-initiation complex or replication forks. Such molecules may for example include helicases such as eukaryotic helicase or MCM proteins (MCM2, MCM3, MCM4, MCM5, MCM6, MCM7), a TP protein as set forth in WO0050451 and WO0217947 (also referred to as HELAD1, Pomfil2, Unc-53) , kinases or phosphatases involved in the replication process such as, for example, CDC6, CDC7 protein kinase, Dbf4, CDC14 protein phosphatase, CDC45 and MCM10. In addition, proliferation markers can include proteins that are part of the replication fork, such as e.g. PCNA or delta DNA polymerase, replication protein A (RPA), replication factor C, FEN1.

In other embodiments, proliferation markers may include molecules necessary to maintain cell proliferation, such as Ki67 or KiS2. In this embodiment, for example, proteins may be present throughout the entire cell cycle. They are useful for testing in accordance with the present method provided that they are characteristic of active cell proliferation and are not produced in appreciable amounts in a proliferation arrested state, in terminally differentiated cells, in a state of apoptosis or senescence. Ki67, Ki-S2 and Ki-S5 as used herein will denote the marker protein molecules detected by the respective antibodies as well as the nucleic acids encoding these antigens.

In another embodiment, the cell proliferation marker for use in the assay according to the present method may be a delayed or terminated proliferative marker molecule.

An aging marker, a cell cycle arrest marker, a marker that is characteristic of terminally differentiated cells, or an apoptosis marker. Such molecules include e.g. p21, p27, caspases, BAD, CD95, fas ligand, parp proteins etc.

The distinction used in the context of this method will include the assessment of whether a sample should be classified in one way or another. In one variation of the method, the distinction is made in assessing whether the tissue or its components are dysplastic or non-dysplastic. Thus, the distinction used herein relates to the growth properties of the cells in the biological sample.

In one embodiment of the present method, the discrimination comprises detecting the expression of the INK4a gene product simultaneously with detecting the expression of a marker characteristic of active cell proliferation. In this case, cells expressing both marker particles simultaneously should be classified as dysplastic.

In another embodiment of the present method, the discrimination comprises the detection of the INK4a gene product and the simultaneous detection of the expression of a marker characteristic of the arrest, termination or delay of cell proliferation. In this case, cells showing the simultaneous expression of both marker particles should be classified as non-dysplastic.

In some embodiments of the present method, it will be useful to detect the presence or absence and / or the level of more than two marker particles. In one embodiment, one INK4a gene expression product will be detected in conjunction with two or more cell proliferation markers. This may be useful for better identification of the proliferative properties of cells expressing the INK4a gene product in samples. Some markers of proliferation are restricted to specific phases of the cell cycle or are present in low numbers in cells. Therefore, in some cases, the detection of proliferating cells expressing the INK4a gene products can be improved by detecting two or more proliferation markers. In such cases, for example, when one of the proliferation markers is present throughout the cell's proliferation cycle, it may be detected simultaneously with markers characteristic of specific phases of the cell cycle. For example, Ki67, Ki-S5 or Ki-S2 can be detected simultaneously with mcm5, mcm2, PCNA, RPA, rfC etc. In other cases, e.g. proteins involved in the DNA replication process can be detected simultaneously with Ki67, KiS5 or Ki -S2. In yet another case, Ki67 can be detected together with Ki-S2. It should be understood that these examples are intended to illustrate the combination possibilities and are not exhaustive, so that the use of various other combinations of proliferation markers is equally useful and appropriate in the assay procedure of the invention.

There may be instances where a combination of two or more cell proliferation markers may be used in the method described herein. In another embodiment, two or more marker molecules detectable throughout a long part of the cell cycle or even throughout the cell cycle or in actively proliferating cells may be detected simultaneously as described herein. The combination of the detection of more than one cell proliferation marker molecule may be generally useful for increasing the sensitivity of detecting cell proliferation characteristics.

In some embodiments of the present method, the combination may also include other marker molecules such as cell aging marker molecules, cell proliferation arrest markers, terminally differentiated cell markers, markers of apoptotic cells, markers of viral infection or viral activity in cells, or regulatory protein markers. cell cycle. In some embodiments, due to the association of dysplasia with HPV infections, detection of HPV-related marker particles or detection of markers of viral activity may be used to detect dysplasia. Methods useful for detecting HPV infection in samples are known to those of skill in the art. These methods include HPV factor specific probe assays and may include nucleic acid amplification reactions. Detection of viral infection can be performed simultaneously with detection of INK4a and the proliferation marker particles or at a later time.

The term dysplastic as used in the context of the present method refers to the mild to severe forms of dysplasia and their precursor stages, as well as to neoplasms such as in situ neoplasms or invasive neoplasms and disseminated neoplastic cells. Therefore, as used herein, the term dysplastic will also include the early and precursor stages of dysplasia and neoplasms.

Cells that overexpress INK4a gene products that are not dysplastic (the term non-dysplastic as used herein) may include metaplastic cells, senescent cells, terminally differentiated cells, or cells that exhibit at certain stages of the cell cycle

Increased concentrations of INK4a gene products. In some cells, elevated concentrations of INK4a gene products may occur in response to external signals such as hormones, messengers, etc. In one embodiment of the present method, non-dysplastic cells overexpressing INK4a gene products include metaplastic cells, endometrial cells, etc.

A suitable method for detecting the expression level of the INK4a gene products and / or the proliferation marker gene products is any method which may or may not be suitable for detecting even very small amounts of specific biological particles in biological samples. The detection reaction of the present method is detection either at the nucleic acid level or at the protein level.

In the context of this method, a marker molecule is said to be detectable if it can be detected by appropriate detection procedures such as e.g. in-situ hybridization, immunochemical staining, hybrid capture assay, etc. the level of expression of the marker molecule will be detectable using an appropriate reporter reaction such as e.g. chromogenic or fluorescent immunochemical staining or in-situ hybridization method for microscopic or automated analysis. Any suitable methods for increasing the reporter signal known to those skilled in the art can be used in this method.

Therefore, a marker is said to be detectable when the staining displaces the corresponding background staining obtained by the immunochemical staining procedure giving significant staining results.

Marker particles can be detected using reagents that specifically recognize the particles. The detection reaction of the INK4a gene products and / or the proliferation marker gene products may involve one or more reactions with detection agents that recognize the initial marker molecules or molecules previously used to recognize other particles.

In some embodiments of the present method, two or more probes may be used to detect a single marker molecule. For example, two or more different binding agents (e.g., antibodies) or oligonucleotide probes directed against a single marker moiety (as the case may be directed against different epitopes or different sequences) can be used.

Detection of different gene products can be performed in one reaction vessel or container or in different containers simultaneously or sequentially over time. In this way, different gene products can be detected simultaneously in one cell expressing both products. Other cells simultaneously expressing the gene products can be used in separate detection reactions (spaced or spaced apart) to detect each individual marker in the cells. In another embodiment, cells expressing one or the other marker may be used. Detection of a marker molecule in different cells can also be performed simultaneously or separately in time and / or space.

The detection reaction further may comprise a reporter reaction indicative of the presence or absence and / or the level of the marker molecule gene products. The reporter reaction can be, for example, a color compound reaction, a bioluminescence reaction, a fluorescence reaction, or a radiation emitting reaction in general, etc.

In some embodiments, different marker molecules can be recognized by agents that produce different reporter signals such that signals pertaining to the marker particles can be distinguished. In one particular embodiment of the method, the detection of the expression of two or more INK4a gene products and / or the proliferation marker gene products is performed simultaneously. In this case, the reporter reaction may, for example, use different fluorescent labels for the different detected particles.

However, in the context of this method, it is not necessary to ascertain whether one or more of the proliferation marker or the marker gene product INK4a is produced in the cell. In some embodiments, the question is whether any proliferation marker and / or INK4a gene product is being produced. In the course of the experiments, one can choose a procedure that gives the same fluorescence signal indicating the presence of a proliferation marker. This procedure is suitable for improving the sensitivity of detecting the features of cell proliferation (various markers characteristic of active cell proliferation). Depending on the circumstances, a procedure yielding one detectable signal for three, four or even more marker particles characteristic of cell proliferation may be used. Likewise, the same may under certain circumstances be true of the INK4a gene expression products. It should be understood that, depending on the circumstances, an application may be desirable

Different staining signals for different proliferation marker particles. Procedures may be used depending on the needs of the relevant experiment.

In some embodiments of the present method, the combination of one or more (e.g., two different) INK4a gene products may be detected in combination with one or more, e.g., a set of two, a set of three, a set of four, a set of five, or even a greater set of cell proliferation markers. Depending on the circumstances, detection of cell proliferation marker particles may yield only one reporter signal. In other instances, each individual marker of cellular proliferation may give a specific reporter signal, or groups of marker particles may give specific reporter signals.

Signals indicative of immunoreactivity may be chromogenic signals produced in a variety of ways. Alternatively, or even in combination, fluorescent signals can be used. Suitable reporter signals include fluorescent labels such as fluorescein, rhodamine, etc.

Suitable types of detection reactions according to the present method may be blotting techniques such as Western-Blot, Southern-blot, Northem-blot, immuno-cytochemical or immuno-histochemical procedures. Blotting techniques are known to those skilled in the art and can be performed, for example, as electroblotings, semi-dry blots, vacuum blots or dot blots. Immuno-cyto / histochemical staining procedures are known to those skilled in the art and may include detection of polypeptides using a binding agent and in situ hybridization techniques. Both of these techniques can even be used simultaneously. In some embodiments, capture of nucleic acid hybrids may be used for detection. The amplification reaction may also be suitable for the detection of, e.g., nucleic acid particles.

In one embodiment of the invention, the detection of the level of INK4a and / or the detection of the proliferation marker gene products is performed by detecting the corresponding nucleic acids (e.g. mRNA) or fragments thereof present in the sample. Means for detecting nucleic acid particles are known to those skilled in the art. The procedure for detecting nucleic acids can also be carried out, for example, by a binding reaction of the molecule to be detected to complementary nucleic acid probes, nucleic acid-specific binding proteins or any other entity specifically recognizing and binding nucleic acids. In one embodiment, in situ hybridization of oligonucleotide probes to nucleic acids can be used to detect expression products or markers.

The probes used in the context of this method can be binding agents specific to a molecule. In the case of nucleic acids, the probe may be an oligonucleotide that hybridizes to the appropriate sequence. In one embodiment, the probe may be, e.g., a primer. When detecting polypeptides or proteins, the probe used can be, for example, a binding agent such as an antibody. In some embodiments of the present method, the probes may be labeled such that they can be detected. The label can be selected from the group consisting of a radioisotope, a bioluminescent compound, a chemiluminescent compound, a fluorescent compound, a chelating metal or an enzyme. The probes can be used in any known detection procedure, e.g., in situ hybridization, hybrid capture, immunochemical staining, blot techniques, etc.

This method can be performed both in vitro and in situ, for example in detecting a staining reaction. Another method of detecting a marker mRNA in a sample made in accordance with the present method is a nucleic acid amplification reaction that can be performed quantitatively, such as, for example, a polymerase chain reaction (PCR). In a preferred embodiment of the present method, Real Time PCR (Real Time PCR) can be used to quantify the concentration of marker mRNA in samples of dysplasia or neoplastic lesions (cells or tissue samples).

In another preferred embodiment of the method, the detection of the level of INK4a and / or the proliferation marker gene products is performed by determining the expression level of the protein or fragments thereof. The determination of the marker gene product at the protein level can, for example, be carried out in a reaction involving a binding agent specific for the detection of a given marker polypeptide.

Binding agents can be used in a wide variety of techniques such as, for example, Western blot, ELISA, or immunoprecipitation. Typically, detection based on the detection of the polypeptide binding agent can be performed both in vitro and directly in situ, for example by an immunochemical staining reaction. Any other test for quantifying the amount of specific polypeptides in biological samples can also be used in the present method.

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Immuno-cytochemical imaging procedures used in the context of the present method may include, for example, staining of cytological or histological slides with chromogenic or fluorescent dyes. The staining may e.g. involve the binding of particles to be detected by a primary binding agent that is itself recognized by a secondary binding agent that can be labeled. The primary binding agent may be a nucleic acid or protein (e.g., an antibody) binding agent in some embodiments, and the secondary binding agent may be, e.g., a secondary antibody that recognizes a primary binding agent.

Any known cytochemical or histochemical staining method can be used in this procedure.

Binding agents used in the context of this method to detect the level of INK4a polypeptides such as p16^{! NK4a} or p14ARF polypeptides and proliferation marker polypeptides such as mcm5, mcm2, KI67, Ki-S5, PCNA or Ki-S2 polypeptides may include antibodies and antigen binding fragments, bifunctional hybrid antibodies, peptidomimetics containing minimal antigen binding epitopes, caline ™), etc.

An antibody or antigen binding agent is considered to react specifically if it reacts at a detectable level with the specific protein and does not substantially react with other proteins. The antibodies that can be used in the present invention can be monoclonal or polyclonal antibodies. The term antibody or monoclonal antibody as used herein denotes intact antibody molecules and fragments. In addition, the antibodies that may find use in the present invention include chimeric, single chain, and humanized antibodies.

In this method, the binding agents can be used individually or in combination. By combining it is possible to obtain a higher degree of sensitivity. The term antibody refers to antibodies that primarily include pooled monoclonal antibodies with different epitope specificities, as well as discrete preparations of monoclonal antibodies.

Monoclonal antibodies are produced from an antigen containing fragments of a polypeptide described in the present method by any variety of techniques known to those of skill in the art; see, e.g., Harlow and Lane, Antibodies: A Laboratory manual, Cold Spring Harbor Laboratory, 1988. In one such technique, an immunogen comprising an antigenic polypeptide or a synthetic portion thereof is initially injected into a variety of mammals (e.g., mice, rats, rabbits, sheep, and goats). At this stage, the polypeptide in the method can serve as an immunogen without modification. Alternatively, especially for relatively short polypeptides, a higher immune response may be elicited if the polypeptide is linked to a carrier protein such as bovine albumin or keyhole limpet hemocyanin. Immunogen is injected into the host organism according to a predetermined regimen of one or more booster immunizations and blood is taken periodically. The polyclonal antibodies specific for the polypeptide can then be purified from such antiserum by, for example, affinity chromatography, using the polypeptide coupled to a suitable support solid.

In the context of this method, it is not necessary to answer the question whether one or more proliferation markers are produced in cells. In some embodiments, the main question may be whether any proliferation marker is being produced. Therefore, in the course of the experiments a procedure was chosen that gives the same fluorescent signal which is an indicator of the presence of a proliferation marker. This procedure is suitable for improving the sensitivity of detecting the features of cell proliferation. Depending on the circumstances, a procedure giving one detectable signal to three, four or even more marker particles characteristic of cell proliferation may be used. Likewise, the same may under certain circumstances be true of the INK4a gene expression products. It should be understood that depending on the circumstances, it may be desirable to use different staining signals for different proliferation marker particles. Different procedures can be used depending on the needs of the experiment.

The INK4a gene products and / or the proliferation marker gene products can be detected simultaneously in this method. In this context, simultaneously means either literally at the same time or within a single test procedure, where the individual detection steps follow one another over time.

The detection procedure according to the present method may further include a cytochemical staining procedure that produces chromogenic or fluorescent staining of cells or cell divisions. Such staining procedures are known to those of skill in the art and can

These include, e.g., staining of eosinophilic or basophilic structures, various regions within a cell (e.g., nucleus, mitochondria, Golgi apparatus, cytoplasm, etc.), specific particles (chromosomes, lipids, glycoproteins, polysaccharides, etc.) in samples cytological. Fluorescent dyes such as DAPI, Quinacrin, Chromomycin, etc. can be used. In addition, fluorescent dyes such as Alan, acridine orange, hematoxylin, eosin, Sudan red, thiazine dyes (toluidine blue, thionine) can be used. In other embodiments of this method, staining procedures such as Pap staining, Giemz staining, hematoxylin-eosin staining, van-Gieson staining, Schiff staining (with Schiff reagent), metal precipitation staining procedures (such as e.g. silver in silver nitrate dyeing procedures) or insoluble dyes such as e.g. Tumbulls blue (or other insoluble metal cyanides), etc. It should be understood that the dyes and dyeing methods mentioned are only examples and that any other dyes may be used in this procedure known method.

Staining procedures may use chromogenic dyes for light microscopy evaluation or fluorescent dyes for fluorescence microscopy evaluation. In another embodiment of this method, radiation-producing procedures, procedures that use radiation-interfering substances, or other contrast agents to visualize cytological conditions in the sample (e.g. the formation of optical reflection in the process of creating a (micro) autoradiographic or (micro) radiographic image).

All staining and imaging procedures can be used for analysis not only in microscopic procedures, but also in automated analysis procedures such as flow cytometry, automated microscopic analysis (computerized or computer assisted) or any other method for analyzing stained cytology samples.

The analysis of the results of the staining or imaging of the various procedures may be performed in one step or in a series of consecutive steps. For example, analysis of a sample under a light microscope may be performed before or after analysis of the sample under a fluorescence microscope. In fluorescence microscopy, the analysis of different dyes that excite different wavelengths of light can be performed simultaneously or in steps. Other imaging methods may be used concurrently with or following these procedures.

There may be different circumstances in which a combination of different staining methods may be appropriate. For example, in cases where adequate cytological staining cannot be obtained by immunochemical staining, the additional use of general cytological staining techniques may be appropriate.

The sample in this method can be any sample that contains cells. The samples can be, for example, secretions, swabs, body fluids, and cell and tissue samples.

In one embodiment of the present method, the samples include cells of the genitourinary, respiratory, or skin and its appendages. In one embodiment, the cells may be from the cervix, vagina, vulva, penis, anus, rectum, bronchial tree, lung, peritoneum, peritoneal space, nasopharynx, mouth, or skin. In some embodiments of the present method, a sample may be a biological sample, a biopsy sample, or a cytological sample, such as, e.g. smear, swab, washings, body fluid containing cells (sputum, secretions, saliva, etc.). In some embodiments of the present method, the samples may include cells infected with papillomavirus. The samples may in some embodiments include cervical smears, bronchial washes, etc.

In certain particular embodiments of the present method, the sample may be prepared as a monolayer or thin layer preparation of a cytological sample. Suitable methods for the preparation of a monolayer or a thin layer cytological preparation are known to those skilled in the art. In one embodiment, the formulation may, for example, include ThinPrep technology. Other methods include conventional smears or methods using cell suspensions to prepare cytological samples.

Sample preparation may include, e.g., obtaining a sample from tissue, body fluid, patient cells. In accordance with this method, sample preparation may also include several steps of further sample preparation, such as preparation of slides, preparation of a cell suspension, smearing or application of cells to be tested on microscope slides, preparation of tissue probes, isolation of polypeptides or nucleic acids, phase preparation constant of fixed peptides or nucleic acids or preparation of beads, membranes or slides, to which the molecules to be identified are covalently and non-covalently bound.

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In some implementations of this method, it may be performed automatically. Automation of the method can be achieved by automatic staining and analysis of histological or cytological samples on a solid support using a microscope. In another embodiment, the automation may include analyzing stained cells by solution flow cytometry.

Dysplastic changes to which this method can be applied are all dysplastic changes characterized by overexpression of INK4a gene products such as e.g., p16^INK4aor p14ARF. In some embodiments, the changes are dysplasia associated with infections caused by papillomaviruses such as e.g. HPV. In one embodiment, it may be a high risk variant of HPV, such as HPV 16, HPV 18, HPV 31, HPV 33, HPV 35, HPV 39, HPV 45, HPV 51, HPV 52, HPV 56, HPV 58, HPV. 59, HPV 66, HPV 68, etc. In another embodiment, dysplastic changes that can be detected according to the present method include anorectal changes, changes in the respiratory system, changes in the head and neck, or changes in the skin and its appendages. Such changes may include, for example, dysplasia of the anus or rectum, vulva, vagina, cervix or penis, bronchial tree, lung, mouth, nasopharynx.

Another aspect of this method is the test kit that is used to perform the test as per the procedure. This kit can be a diagnostic or research kit.

The kit according to the present invention comprises at least one agent suitable for the detection of the INK4a gene products.

Thus, the kit used in this method may include reagents for detecting one or more INK4a gene products, reagents for detecting one or more proliferation marker gene products, reagents and buffers commonly used to perform detection reactions, such as buffers, reporter reagents (dyes, etc.) , carrier substances and other samples of the INK4a gene product to perform the positive control reaction a sample of the proliferation marker gene product to perform the positive control reaction.

Reagents for detecting the marker gene products may include any agent that binds to the marker gene products. Such reagents may include proteins, polypeptides, nucleic acids, peptide-linked nucleic acids, glycoproteins, proteoglycans, polysaccharides, or lipids.

The samples of the INK4a gene product and the proliferation marker gene product to be used for positive control may contain, for example, nucleic acids in a suitable form such as solution or salt, peptides in an appropriate form, samples of tissue or cells expressing the gene of interest.

In a preferred embodiment of the method, the detection of the marker gene products is performed at the polypeptide level. In this embodiment, the binding members can be, for example, antibodies specific for the marker gene products or fragments thereof.

In another embodiment of the test kit, detection of the marker gene products is performed at the nucleic acid level. In this embodiment of the method, the detection reagent may be, for example, a nucleic acid probe or a primer complementary to the nucleic acid of the marker in question.

The present invention relates to a method of diagnosing neoplastic and / or dysplastic and pre-neoplastic lesions, identifiable by assessing the level of p16 overexpression.^INK4a, in cells that also express p16^INK4aat a level that is detectable by histological and / or cytological tests. The method is based on the detection of the expressed gene products of two or more INK4a gene products.

Accordingly, the main problem that would need to be solved was to provide a method to distinguish between dysplastic cells and other cells that do not have a malignant growth potential. The method can be used in all degrees of dysplasia and can be particularly useful in the early stages when cytological diagnostic methods based on p16 overexpression^INK4a require additional information to identify metaplastic cells.

In addition, this method provides a kit to perform the test as per the procedure.

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Brief description of the drawings

Figure 1: Fluorescent staining of the cervical histology sample;

Figure 1 shows staining of severe dysplasia with antibodies directed against p16^INK4a. Details of the procedure can be found in Example 1. p16 immunoreactivity^INK4a gives green fluorescence. Almost all cells within the lesion are stained in a diffuse manner within the cytoplasm and in the nucleus of the cells.

Figure 2: Fluorescence staining of the cervical histology sample;

Figure 2 shows staining of severe dysplasia with antibodies directed against Ki67. Details of the procedure can be found in Example 1. Immunoreactivity against Ki67 gives red fluorescence. Many cells in the dysplasia show nuclear staining for Ki67.

Figure 3: Double fluorescence staining of the cervical histology sample;

Figure 3 shows staining of severe dysplasia with anti-Ki67 and anti-p16 antibodies^INK4a. Details of the procedure can be found in Example 1. Immunoreactivity against Ki67 gives red fluorescence; p16 immunoreactivity^INK4a gives green fluorescence; overlaying red and green fluorescence gives yellow fluorescence. Many cells within the dysplasia show nuclear staining for Ki67 as well as staining for p16^INK4a and therefore gives yellow fluorescence.

Figure 4: Fluorescent staining of the cervical histology sample;

Figure 4 shows staining of a squamous metaplasia with antibodies directed against p16^INK4a. Details of the procedure can be found in Example 1. p16 immunoreactivity^INK4a gives green fluorescence. Some cells of the lesion are diffused stained within the cytoplasm and in the nucleus.

Figure 5: Fluorescent staining of the cervical histology sample;

Figure 5 shows staining of a squamous metaplasia with antibodies directed against Ki67. Details of the procedure can be found in Example 1. Immunoreactivity against Ki67 gives red fluorescence. Many cells in the squamous metaplasia show nuclear staining for Ki67. Areas where p16 expression has been demonstrated^INK4a they show no staining for Ki67, indicating no antigen expression in these areas.

Figure 6: Double fluorescence staining of the cervical histology sample;

Figure 6 shows the staining of a squamous metaplasia with anti-Ki67 and anti-p16 antibodies.^INK4a; details of the experiment can be found in Example 1. Immunoreactivity against Ki67 gives red fluorescence, immunoreactivity against p16^INK4a gives green fluorescence; overlaying red and green fluorescence gives yellow fluorescence. Areas that express p16^INK4a they show no staining for Ki67, indicating no antigen expression in these areas. None of the sample cells showed yellow fluorescence.

Figure 7: Double chromogenic staining of the cervical histology sample;

Figure 7 shows staining of severe dysplasia with anti-Ki67 and anti-p16 antibodies.^INK4a; details of the experiment can be found in Example 6. Immunoreactivity against Ki67 gives red colored nuclei, immunoreactivity against p16^INK4a gives a brownish color to whole cells; double staining results in brown cells with red cell nuclei. Many cells within the dysplasia show nuclear staining for Ki67 as well as positive p16 staining^INK4a and therefore they give the image of brown cells with red nuclei.

Figure 8: Double chromogenic staining of the cervix cytology sample;

Figure 8 shows staining of severe dysplasia with anti-Ki67 and anti-p16 antibodies.^INK4a; details of the experiment can be found in Example 7. Immunoreactivity against Ki67 causes red staining of the testes, immunoreactivity against p16^INK4a gives a brownish color to whole cells; double staining results in brown cells with red nuclei. Many cells within the dysplasia show nuclear staining for Ki67 as well as positive p16 staining^INK4a and therefore they give the image of brown cells with red nuclei.

The following examples are provided for illustrative purposes only and are not intended to limit the purpose of the method presented herein. For illustrative purposes, the methods were presented using histological slides. Histological examples help to judge whether

Cells stained in one way or another should be classified as dysplastic or metaplastic. These methods can be easily transferred to cytological samples by changing the protocol as appropriate. These changes are known to those skilled in the art.

Example 1: Detection of p16 overexpression^INK4a and Ki-67 in cervical specimens by immunofluorescence (double staining)

Formalin-fixed and paraffin-embedded cervical sections were immunofluorescent stained with p16-specific antibodies^INK4aand Ki67.

Tissue sections were rehydrated by incubation in xylene and increasing concentration of ethyl alcohol and transferred to Aqua bidest. Antigen searches were performed in 10 mM citrate buffer (pH 6.0) for p16^INK4a and Ki67. For this, the sections were heated in a water bath for 40 min at 95 ° C - 98 ° C, then cooled to room temperature for 20 minutes, transferred to the washing buffer (50 mM Tris-HCl, 150 mM NaCl, 0.05% Tweed 20 / DakoCytomation: Code No: S3006).

To avoid non-specific binding of secondary antibodies (goat species), samples were incubated with 10% goat serum for 30 min at room temperature.

Sections were incubated with primary antibodies, mouse antibodies directed against human p16^INK4a (3.48 μg / ml) and rabbit anti-Ki67 antibodies (1:25) for 30 min at room temperature, then rinsed with washing buffer and placed in fresh solution for 5 min. Excess buffer was poured off and each sample was primed with 200 μl of secondary reagent containing goat antimouse antibody conjugated to AlexaFluor®488 and goat anti rabbit antibody conjugated to AlexaFluor®546 and incubated with AlexaFluor®546. 30 min at room temperature. Then, sections were washed twice as before and mounted on a slide with special mounting medium for fluorescence.

Microscopic examination of the slides shows that the cells are immunoreactive with p16^INK4a and for Ki67 can only be found in samples that can be identified microscopically as samples of dysplastic lesions. Cells stained with a p16 specific reaction^INK4athose derived from metaplastic changes do not stain in the Ki67 specific reaction. Microscopic evaluation of the cell proliferation marker staining shows that metaplastic cells overexpress p16^INK4a and are not immunoreactive with antibodies directed against Ki67. In contrast, samples containing areas of dysplastic tissue contain cells that are immunoreactive against Ki67 and against antibodies directed against p16^INK4a. Thus, unlike dysplasia, in metaplasia no cells show double staining with antibodies against Ki67 and p16^INK4a.

Figures 1-6 show the results of staining of cervical tissues showing severe dysplasia and squamous metaplasia with anti-Ki67 and anti-p16 antibodies.^INK4a. Ki67 immunoreactivity gives red fluorescence, p16 immunoreactivity^INK4a gives green fluorescence, and overlaying red and green fluorescence gives yellow fluorescence. In the dysplastic sample (Fig. 1-3), many dysplasia cells show nuclear staining for Ki67 as well as for p16^INK4a and therefore gives yellow fluorescence (see Figure 3). In contrast, in the metaplastic sample (Figs. 4-6) areas showing expression of p16^INK4a they show no staining for Ki67, indicating no antigen expression in these areas. Double staining can be observed in this sample (Fig. 6) and therefore no cells in the sample show yellow fluorescence.

The results indicate that double staining of the cells with Ki67 specific reagents allows the discrimination of p16 overexpression^INK4a in metaplastic and dysplastic changes.

Example 2: Detection of cells expressing p14ARF and mcm2 simultaneously in cervical samples by in-situ hybridization

Cervical smears can be analyzed semi-quantitatively for p16 mRNA levels^INK4a and mcm2 by in-situ staining. The staining reaction is performed as follows:

For rehydration, the spray-fixed smears are incubated in fresh 50% ethyl alcohol on a shuttle device. The PEG layer formed during the fixing procedure is removed by intensive washing. Then the smears are rinsed with aqua bidest. The smears are incubated with proteinase K (10 µg / ml in PBS) for 10 min at 37 ° C. Then the preparations were processed

The parts are brought into the washing buffer (PBS / 0.1% Tweed 20) and finally the area containing the cells is surrounded with a lipid-pencil. The hybridization mixture is prepared by mixing 50 µΐ of ready-to-use hybridization buffer (DAKO A / S, Glostrup, Denmark) and approximately 5-10 pmol of probes. The probes are biotin and digoxigenin labeled oligonucleotides with sequences complementary to the corresponding mRNAs.

The hybridization mixture is heated to 95 ° C and then cooled to 37 ° C. Following the cooking procedure, smears are incubated with 50 µΐ of hybridization mixture for 4 hours at 42 ° C. Samples are washed in excess of the washing buffer volume twice in 2 x SSC at 37 ° C for 15 min and once in 1 x SSC at 37 ° C for 15 min. Then the smears are rinsed twice at room temperature in 2 x SSC. Following this washing procedure, slides are incubated for 30 min with blocking buffer (NEN, Blockingbuffer) at room temperature. They are then incubated for 1 hour with 1: 100 diluted (in blocking solution, see above) alkaline phosphatase-streptavidin complex and anti-digoxigenin-labeled monoclonal HRP antibodies (Molecular Probes). The smears are then washed sequentially 2 times in 1 x PBS / 0.1% Triton X-100 for 10 min at room temperature and once in 1 x PBS, 50 mM MgCl₂ (pH 9.2) for 10 min at room temperature. This is followed by a staining reaction with ELF 97 phosphate (Molecular Probes) for 10 seconds to 7 minutes at room temperature. Excess substrate is washed 3 times with 1 x PBS / 0.1% Triton X-100 for 10 min at room temperature. In the second staining step, slides are incubated in Tyramides-Alexa-Fluor 594 for 10 seconds to 7 minutes. Excess substrate is washed 3 times with 1 x PBS / 0.1% Triton X-100 for 10 min at room temperature. Finally, the smears are immersed in distilled H2O and embedded in fluorescence mounting medium (DakoCytomation). The stained slides can then be assessed by fluorescence microscopy.

Microscopic examination of the preparations shows that the cells expressing p16^INK4a and mcm2 can only be found in samples that can be identified microscopically as samples of dysplastic lesions. Cells stained by p16 specific reaction^{! NK4a}, which can be identified as metaplasia, do not stain in the mcm2-specific reaction. Microscopic evaluation of mRNA hybridization shows that metaplastic cells overexpressing p16^INK4a do not significantly express mcm2 mRNA. Dysplastic cells, on the other hand, can be stained by in-situ hybridization with probes specific for mcm2 and probes directed against p 1 6^INK4a . Thus, in contrast to dysplastic cells, metaplastic cells do not show double staining with probes specific for Ki67 and p 1 6^INK4a .

The results indicate that the double staining of cells with mcm2 specific reagents allows the discrimination of metaplastic changes that overexpress p16^INK4a from dysplastic changes.

Example 3: Detection of p16 overexpression^INK4a and Ki-S2 in cervical specimens by immunofluorescence (double staining)

Cervical cytology specimens fixed with Merckofix® (typical smears and cytological preparations made using the ThinPreps® method) were immunofluorescent stained with p 1 specific antibodies 6^INK4a and Ki-S2.

Typical smears and cytological samples made using the ThinPreps® method were hydrated in ethanol (50%) for 10 min and transferred to Aqua bidest. Antigen searches were performed in 10 mM citrate buffer (pH 6.0) for p16^INK4a and Ki67. For this purpose, the preparations were heated in a water bath for 40 min at 95 ° C - 98 ° C and then cooled to room temperature over 20 minutes. The preparations were then transferred to a washing solution (50 mM Tris-HCl, 150 mM NaCl, 0.05% Tweed 20 / DakoCytomation: Code No: S3006) and finally the samples were circled with a lipid-pencil.

To avoid non-specific binding of secondary antibodies (goat species), samples were incubated with 10% goat serum for 30 minutes at room temperature.

Slides were then incubated with primary antibodies, mouse antibodies directed against human p16^INK4a (clone E6H4) (3.48 µg / ml) and rabbit antibodies against Ki-S2 (1:25) for 30 minutes at room temperature, then the slides were washed with washing buffer and placed in fresh solution for 5 minutes. Excess buffer was poured off and each sample was primed with 200 μl of secondary reagent containing goat anti-mouse antibody conjugated to AlexaFluor® 488 and goat antibodies.

Goat anti rabbit antibody conjugated to AlexaFluor®546 and incubated for 30 minutes at room temperature. Then slides were washed twice as before and mounted on a slide with special mounting medium for fluorescence.

Microscopic examination of the slides shows that the cells exhibiting p16 immunoreactivity^INK4a and to Ki-S2 can be identified microscopically as dysplastic cells. Cells stained with a p16 specific reaction^INK4athat do not stain by the Ki-S2 specific reaction can be classified by the experienced pathologist as either metaplastic or endometrial in origin. Microscopic evaluation of the cell proliferation marker staining shows that metaplastic cells overexpressing p16^INK4a they are not immunoreactive with antibodies directed against Ki-S2. Dysplastic cells are immunoreactive against Ki-S2 and with antibodies directed against p16^INK4a. Thus, unlike dysplastic cells, in metaplasia no cells show double staining with antibodies against Ki67 and p16^INK4a.

These results show that double staining of cells with Ki-S2 specific reagents allows the discrimination of p16 overexpressing metaplastic cells.^INK4a from dysplastic cells.

Example 4: Detection of p16 overexpression^INK4a, Ki67 and PCNA in samples from bronchial washes from people diagnosed with small cell lung cancer by immunofluorescence (double staining)

The cells contained in the bronchial washes were prepared according to the ThinPrep technology. Cytological samples of the lavage from patients diagnosed with small cell lung cancer, fixed with Merckofix, were subjected to immunofluorescence staining with p16 specific antibodies^INK4a, Ki67 and PCNA.

In this experiment, a procedure was used that did not discriminate between staining originating from the immunoreactivity of the two proliferation markers PCNA and Ki67. In the context of this method, it is not necessary to answer the question of whether the cells express any of the markers of proliferation. The main question is whether the cells express any marker. Therefore, in the course of the experiments, a procedure was chosen that gives the same fluorescence signal as evidence of the presence of a proliferation marker. This procedure is used to increase the sensitivity of detecting the features of cellular proliferation. Depending on the circumstances, a procedure may be used that produces one detectable signal for three, four or even more marker particles characteristic of cell proliferation. Likewise, the same may under certain circumstances be true for the expression products of the INK4a gene. It should be understood that depending on the circumstances, it may be desirable to use different staining signals for different proliferation marker particles. The procedures can be used depending on the needs of the relevant experiment. Tissue sections were rehydrated by incubation in xylene and increasing concentration of ethyl alcohol and transferred to Aqua bidest. Typical smears and cytological samples made using the ThinPreps® method were hydrated in ethanol (50%) for 10 minutes and then transferred to Aqua bidest. Antigen searches were performed in 10 mM citrate buffer (pH 6.0) for p16^INK4a, Ki67 and PCNA. For this purpose, the preparations were heated in a water bath for 40 min at 95 ° C - 98 ° C and then cooled to room temperature for 20 minutes; transferred to a washing buffer (50 mM Tris-HCl, 150 mM NaCl, 0.05% Tween 20 / DakoCytomation: Code No .: S3006) and finally the samples were circled in lipid-pencil.

To avoid non-specific binding of secondary antibodies (goat species), samples were incubated with 10% goat serum for 30 minutes at room temperature.

Slides were then incubated with primary antibodies, mouse antibodies directed against human p16^INK4a (3.48 µg / ml), rabbit anti-Ki67 antibodies and rabbit anti-PCNA antibodies (1:25 each), for 30 min at room temperature, then the sections were washed with washing buffer and placed in fresh solution for 5 min. Excess buffer was poured off and each sample was primed with 200 μl of secondary reagent containing goat anti-mouse antibody conjugated to AlexaFluor® 488 and goat anti rabbit antibody conjugated to AlexaFluor®546 and incubated for 30 min at room temperature. Then slides were washed twice as before and mounted on a slide with special mounting medium for fluorescence.

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Microscopic examination of the slides shows that the cells exhibiting p16 immunoreactivity^INK4a and to Ki67 or PCNA can be identified microscopically as small cell lung carcinoma cells. Cells stained with a p16 specific reaction^INK4athose resulting from metaplastic changes do not stain in the specific reaction for Ki67 and PCNA. Microscopic evaluation of the cell proliferation marker staining shows that metaplastic cells overexpressing p16^INK4a they are not immunoreactive with antibodies directed against Ki67 and PCNA. Samples containing dysplastic cells contain cells showing immunoreactivity against Ki67 / PCNA and against antibodies directed against p16^INK4a. Thus, unlike dysplasia, in metaplasia no cells show triple staining with antibodies against Ki67 and PCNA and p16^INK4a.

The results indicate that triple staining of cells with Ki67 / PCNA specific reagents can distinguish between non-dysplastic cells overexpressing p16^INK4a from dysplasia.

Example 5: Detection of dysplastic cells by flow cytometry by simultaneous detection of mcm5 mRNA, p14ARF protein and Ki67 protein in cervical cells

Cytological samples (cell suspension in PBS, pH 7.4) of the cervix were fluorescently stained with p14ARF and Ki-67 specific antibodies and mcm5 oligoprobes and evaluated by FACS three-color fluorescence analysis.

The cells were centrifuged, the supernatant was decanted, and the cells were fixed and permeabilized with Permafix (Ortho Diagnostic, Raitan, NJ, USA) for 1 hour at room temperature. Cells were washed in sterile PBS, pH 7.4, centrifuged and resuspended in 100 ml Permeafix for 1 hour at room temperature. Cells were washed in sterile PBS, pH 7.4, centrifuged and resuspended in sterile PBS. It was then incubated with PE-conjugated anti-p14ARF and PE-Cy5-conjugated anti-Ki67 antibody for 1 hour at + 4 ° C. Cells were washed in sterile PBS, pH 7.4, centrifuged and resuspended in 100 ml Permeafix for 30 minutes at room temperature. Cells were washed in sterile PBS, centrifuged and then washed again in 2x standard salt citrate (standard saline citrate, SSC). After centrifugation, the cell pellet was resuspended in hybridization solution (2x SSC, 30% formamide, sonicated salmon sperm, DNA transfer yeast) containing 500 ng of 5-carboxy-fluorescein double-labeled mcm5 specific oligonucleotide probes. Intracellular hybridization was performed at 42 ° C for 1 hour, followed by washing in 2x SSC, 0.5% Triton X100 and 1x SSC, 0.5% Triton X-100 at 42 ° C. Cells were suspended for analysis in PBS, pH 8.3 and analyzed by flow cytometry (FACScan, Becton Dickinson, IS). 30,000-100,000 gated events were collected for each analysis. Data analysis was performed using CellQuest (Becton Dickinson, IS).

Flow cytometric analysis shows that p14ARF immunoreactive cells and simultaneously Ki-67 immunoreactive and / or mcm5 oligopreactive reactive cells can only be identified in patient samples with cervical dysplastic changes. The samples from women without dysplastic changes showed no simultaneous staining for p14ARF and Ki67 or mcm5. These results indicate that double or triple staining of the cells with reagents specific for Ki67 and / or mcm5 allows the discrimination of p14ARF overexpressing non-dysplastic cells from p14ARF overexpressing dysplastic cells.

Example 6: Immunoenzymatic detection of p16 overexpression^INK4a and Ki67 in histological tests of the cervix (consecutive double staining)

Formalin-fixed fragments, paraffin-embedded cervical tissue samples were subjected to enzyme immuno-dual staining with p16 specific antibodies^INK4aand Ki67.

Tissue sections were rehydrated by incubation in xylene and increasing concentration of ethyl alcohol and transferred to Aqua bidest. Antigen searches were performed in 10 mM citrate buffer (pH 6.0) for p16^INK4a and Ki67. For this purpose, the preparations were heated in a water bath for 40 min at 95 ° C - 98 ° C and then cooled to room temperature for 20 minutes; transferred to washing buffer (DakoCytomation).

Endogenous peroxidase activity was blocked with 3% H 2 O 2 (DakoCytomation) for 5 min at room temperature.

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After washing the slides for 5 min at room temperature, they were incubated for 30 min at room temperature with primary antibodies, mouse antibodies directed against human p16^INK4a (MTM), then washed with washing buffer and placed in fresh buffer for 5 minutes. Excess buffer was tapped off and each sample was primed with 200 µ of secondary reagent (EnVision peroxidase conjugated goat anti-mouse / DakoCytomation) and incubated for 30 min at room temperature. Then, as before, the slides were washed three times. DAB (DakoCytomation) was used to visualize the chromogenic image, incubating the slides with the chromogenic substrate complex for 10 min at room temperature. The reaction was stopped in deionized water and the slides placed in the washing buffer.

After washing the preparations, they were incubated for 30 minutes at room temperature with secondary antibodies, rabbit anti-human pKi67 antibodies (Dianova, clone Ab-3), then washed with washing buffer and placed in fresh buffer for 5 minutes. Excess buffer was tapped off and each sample was flooded with 200 µΐ of secondary reagent (alkaline phosphatase labeled goat anti-rabbit / DakoCytomation) and incubated for 30 min at room temperature. Then, as before, the slides were washed three times. FastRed (BioGenex) was used to visualize the chromogenic image, incubating the slides with the chromogenic substrate complex for 30 min at room temperature. The reaction was stopped in deionized water.

After 2 minutes at room temperature counterstaining with hematoxylin (DakoCytomation), slides were incubated under running water for 10 minutes at room temperature and then mounted on a slide with an aqueous binding medium (Aquatex / MERCK).

Microscopic examination of the slides shows that the cells exhibiting p16 immunoreactivity^INK4a and for Ki67 are found only in samples that were identified by microscopic examination as samples coming from the area of ​​dysplastic changes. Cells stained by p16 specific reaction^INK4athose from metaplastic changes were not stained by the reaction specific for Ki67.

Microscopic evaluation of the cell proliferation marker staining shows that metaplastic cells overexpressing p16^INK4a they are not immunoreactive with antibodies directed against Ki67. In contrast, samples containing areas of dysplastic tissue contain cells that are immunoreactive against Ki67 and against antibodies directed against p16^INK4a. Thus, unlike dysplasia, in metaplasia no cells show double staining with antibodies against Ki67 and p16^INK4a. The results shown in Figure 7 indicate that double staining of cells with reagents specific for Ki67 and p16^INK4a allows to obtain a specific image of double staining also in the methods of chromogenic staining.

Example 7: Immunoenzymatic detection of p16 overexpression^INK4a and Ki67 in cervical cytology tests (consecutive double staining)

Merckofix® fixed cytology (ThinPreps®) derived smears and fluids (conventionally used in cytology)) were double-stained by enzyme immunoassay using p16 specific antibodies^INK4a and Ki67.

The smear and fluid samples conventionally used in cytology were rehydrated in ethyl alcohol (50%) for 10 min at room temperature and transferred to Aqua bidest. Searching for p16 antigens^INK4a and Ki67 was performed in 10 mM citrate buffer (pH 6.0). For this purpose, the preparations were heated in a water bath for 40 min at 95 ° C - 98 ° C and then cooled to room temperature for 20 minutes; transferred to the washing buffer.

Endogenous peroxidase activity was blocked with 3% H 2 O 2 for 5 min at room temperature.

After washing the slides, they were incubated for 30 minutes at room temperature with the primary antibody, the mouse anti-human p16 antibody.^INK4athen washed with washing buffer and placed in fresh buffer for 5 minutes. Excess buffer was tapped off and each sample was flooded with 200 µ of secondary reagent (EnVision goat anti-mouse perosidase conjugated antibody) and incubated for 30 min at room temperature. Then, as before, the slides were washed three times. DAB (DakoCytomation) was used by incubation to visualize the chromogenic picture

Slides with chromogenic substrate complex for 10 min at room temperature. The reaction was stopped in deionized water and the slides placed in the washing buffer.

After washing the preparations, they were incubated for 30 minutes at room temperature with a second primary antibody, rabbit anti-human pKi67 antibody (Dianova, clone Ab-3), then washed with washing buffer and placed in fresh buffer for 5 minutes. Excess buffer was tapped off and each sample was flooded with 200 µΐ of secondary reagent (alkaline phosphatase labeled goat anti-rabbit / DakoCytomation) and incubated for 30 min at room temperature. Then, as before, the slides were washed three times. FastRed (BioGenex) was used to visualize the chromogenic image, incubating the slides with the chromogenic substrate complex for 30 min at room temperature. The reaction was stopped in deionized water.

After 2 minutes at room temperature counterstaining with hematoxylin (DakoCytomation), slides were incubated under running water for 10 minutes at room temperature and then mounted on a slide with an aqueous binding medium (Aquatex / MERCK).

Microscopic examination of the slides shows that the cells exhibiting p16 immunoreactivity^INK4a and to Ki67 can be identified on the basis of their morphology as samples coming from the area of ​​dysplastic lesions. Cells stained by p16 specific reaction^INK4athose from metaplastic changes were not stained by the reaction specific for Ki67.

Microscopic evaluation of the cell proliferation marker staining shows that metaplastic cells overexpressing p16^INK4a they are not immunoreactive with antibodies directed against Ki67. In contrast, dysplastic cells are immunoreactive against antibodies against Ki67 and against antibodies against p16^INK4a. Thus, unlike metaplastic cells, in the case of dysplastic cells, it is possible to double stain single cells with specific antibodies against Ki67 and p16^INK4a.

The results shown in Figure 8 show that double staining of cells with reagents specific for Ki67 and p16^INK4a allows to obtain a specific image of double staining also in the methods of chromogenic staining.