JP2006521104A - Method for early diagnosis of carcinoma and test kit for performing the same - Google Patents

Abstract

The present invention relates to a method for diagnosing a cancer caused by human papillomavirus (human papillomavirus) or related to human papillomavirus or a pre-stage thereof using a human sample. The method includes determining whether a human papillomavirus protein is expressed. The viral protein is the capsid protein, especially the major capsid protein L1. The invention also relates to a test kit for carrying out this method.

Description

The present invention relates to a method for early diagnosis of a carcinoma caused by human papilloma virus (human papilloma virus) or related to human papilloma virus, or its pre-stage. The invention also relates to a test kit for performing the method.

  Since the end of the 1960s, screening programs for various carcinomas have been marketed in Germany. In the case of cervical cancer, this program is based on cell morphological examination according to G. Papanicolaou (Papanicolaou) method called so-called Pap test on cell smear collected from cervix. For this test, cell samples are taken from the woman at regular intervals within the screening process of a given gynecological examination, smeared on a glass slide, and stained. Classify this smear based on cell morphology and correspond to smear without pathological changes, inflammatory changes, mild dysplasia, moderate dysplasia, severe dysplasia, carcinoma in situ or Pap I to V It is called a cancer type (see Non-Patent Document 1). If the Pap test results show pathological changes, collect biopsies and perform histopathology. This test verifies the nature and extent of dysplasia and classifies it as a cervical intraepithelial neoplasia (CIN I-III). The use of the Pap test over the last 30 years has shown that within a group with uniformly severe malignant degeneration, the likelihood of remission leading to recovery decreases as the severity of dysplasia increases.

  So far, no predictions have been made regarding remission or progression behavior in individual preparations.

  Papillomavirus is a small double-stranded DNA virus with a genome of about 8000 base pairs. So far, more than 150 different virus types that are pathogenic to humans are known. Depending on its epithelial tropism, papillomavirus can be applied to the skin and mucosa group (wart formation, cutaneous neoplasia) or the anogenital group (condyloma, anal and cervical cancer).

  In most cases, infection with HPV is asymptomatic. Therefore, latent viral infections must be distinguished microscopically (“subclinically”) or viral diseases with tissue lesions that can be clinically distinguished.

  Some types of papillomaviruses are oncogenic, ie these viral types (eg HPV 16 and 18) can contribute critically to the transformation of epithelial cells. These viruses are represented as high risk groups. In contrast, there is a low risk group of virus types (eg 6 and 11) that are not carcinogenic at all or very little carcinogenic.

  HPV infection is the most frequent sexually transmitted infection in humans. Prevalence data is significantly different and depends on the initial consultant to be tested. After the first sexual contact, the rate of infection first increases naturally. In North America and parts of Europe, the degree of contact transmission of human papillomavirus is greater than 50% in a group of women aged 15-25 years. The rate of infection detected decreases continuously with increasing age. Less than 5% after 50 years.

  Cervical cells are collected, and then Papanicolaou staining of the cells is performed as a screening method when a woman makes a medical examination. This method allows early detection of changes in cell morphology (pre-cancerous stage) that occur during the progression of cervical cancer. This regular test contributed to a continuous reduction in the incidence of cervical cancer. Nevertheless, every year about 6,000 women in Germany suffer from cervical cancer due to various causes.

  Human papillomavirus plays a critical role in the progression of cervical cancer. Therefore, auxiliary diagnostic methods are directed to discovering infections caused by viruses. When molecular biology methods such as PCR or nucleic acid hybridization are used to detect viral nucleic acids, the majority of latent infections are detected because of the extremely high detection sensitivity of these methods. Therefore, detection of DNA that is always positive in HPV infection is not related to the process of virus growth and cannot be used for prognosis. The importance of viral protein synthesis for the process of infection and the prognosis derived therefrom has not been studied so far.

  The following proteins are known to be produced according to the viral life cycle: early proteins (E) 1, E2, E4, E5, E6 and E7, and late proteins (L) 1 and L2 (2) ).

  Capsid protein L1 is synthesized in the cytoplasm of the host cell and then transported into the nucleus of the cell. In its nucleus, capsid proteins interact with the viral DNA to produce mature viruses. Thus, detection of capsid protein L1 makes it possible to determine that the virus is passing through the growth stage. Mature, infectious virus is released from infected cells. In one specific embodiment, the method is based on a monoclonal antibody that specifically recognizes the papillomavirus capsid protein L1. A suitable monoclonal antibody is disclosed in Patent Document 1. The screening test recognizes all papillomaviruses known to date and is therefore suitable for determining whether capsid antigen has been produced and whether the HPV infection is in its growth stage. The high-risk type inspection selectively identifies HPV risk types 16, 18, 33, 35, 39, 45, 51, 56 and 58 (see, for example, Non-Patent Document 3).

Furthermore, L1-specific antibodies can be used to determine individual HPV types, such as HPV 16.
German Patent Publication No. 43 32 596 (A1) HJ Soost, The Munich nomenclature, Recent Results Cancer Res. 133 [1993] pp. 105-111 HR McMurray, D. Nguyen, TF Westbrook, DJ Mc Cance, Biology of human papilllomaviruses, in Int. J. Exp. Pathol. 82 [2001] 15-33 M. Sapp, U. Kraus, C. Volpers, PJF Snij-ders, JMM Walboomers, JE Streeck, Analysis of type-restricted and cross-reactive epitopes on virus-like particles of human papilloma virus type 33 and in infected tissue using monoclonal antibodies to the major capsid protein, in J. Gen. Virol. 75 [1994] 3375-3383

  The present invention relates to the production of human papillomavirus capsid antigen L1 in the case of mild to moderate dysplasia that is high risk HPV DNA-positive, while the absence of HPV envelope protein is associated with disease progression. Based on the observation that they are interrelated. Therefore, the present invention can perform a relatively safe prognosis as to whether malignant degeneration progresses.

  Accordingly, the subject of the present invention is a method for diagnosing a human papillomavirus (carcinoma caused by or related to human papillomavirus) or a human papillomavirus or a pre-stage thereof using a human body sample. It is the above-mentioned method characterized by examining whether the capsid protein of HPV) is expressed.

  The expression “carcinoma and its pre-stage” includes carcinomas of all types and origins associated with human papillomavirus and their pre-stage. For example, the carcinoma can be an anogenital carcinoma, particularly cervical cancer. Special mention is also made of pre-stages such as mild to moderate dysplasia (cervical intraepithelial neoplasia (CIN I-III), etc.).

   The expression “human papillomavirus capsid protein” means all HPV-type capsid proteins, in particular the viral (major) capsid protein L1 and (minor) capsid protein L2.

   The expression “human body sample” includes any human body sample from which capsid proteins can be detected. Examples of such human samples are smear, biopsy, organ puncture fluid, blood, saliva, urine, stool, cerebrospinal fluid, lymph fluid and the like. If testing for cervical cancer is important, it specifically means smears and biopsies.

  The expression “determining capsid formation” includes all methods suitable for determining the formation of capsid antigen, its mRNA, its intermediates or its precursors. This expression clearly means a replication factor that influences and / or regulates the formation of capsid proteins, and also a serum and / or immune response that should be attributed to the capsid antigen.

   Capsid protein expression can be determined at the nucleic acid level and / or at the protein level.

  Immunohistochemical or immunocytochemical staining methods, and also Western blotting, ELISA or immunoprecipitation methods can be used, for example, for detection at the protein level. Antibodies directed against the capsid antigen can also be used. It is advantageous if the antibody is immobilized on a solid support such as a microtiter plate, test strip or latex particles.

  The method of the present invention can diagnose the process of a carcinoma disease at an early stage, that is, at an earlier stage.

  Another subject of the present invention is a test kit for detecting carcinoma or its pre-stage, which comprises a reagent for examining whether the HPV capsid protein is expressed.

  In this case, the capsid protein generally acts as an antigen. The test kit preferably contains anti-mouse immunoglobulin as a test reagent in combination with an enzyme, preferably peroxidase. In addition, the test kit contains substances for staining the preparation, preferably a chromogen solution, and in addition to this it can contain the usual components such as buffers, carriers and / or markers.

  According to the present invention, carcinoma can be diagnosed at an early stage. In particular, the pre-stage of carcinoma can be examined at an early stage and the course of the disease can be determined.

  It is also characterized in that the results obtained by the method of the invention are not subjectively determined. The present invention is excellent in terms of quick and easy operation. This makes it suitable for large-scale screening methods, especially in third world countries. The present invention thus represents an important contribution to the current diagnosis of HPV-related cancer diseases.

  The invention is illustrated by the following examples.

  A group of 86 cell smears (all of which show mild to moderate dysplasia, and all of these are high-risk HPV DNA-positive) were formed using the L1-specific antibody to form capsid antigen L1 Inspect for. This test is performed using a Viroactiv kit® (available from Virofem Diagnostik und Forschungs [diagnosis and research] GmbH) containing antibodies directed against capsid protein L1. The preparation is boiled and then incubated for 30 minutes at room temperature after addition of antigen. Positive cell nuclei are stained by sequential addition of test reagents (goat anti-mouse immunoglobulin) and chromogen (AEC) solution.

  A smear is determined to be positive if at least one of the cells of the sample shows a specific red staining of the nucleus.

  On this scale, 29 samples were positive. This corresponds to 33.7%. The frequency of remission was 77.3% in women aged 25 years and older with capsid antigen formation. On the other hand, it was about 82.4% of women over 30 years old. The probability of progression to histologically confirmed severe dysplasia or carcinoma in situ was only 22.7% and 17.6%, respectively.

  57 samples were negative for the formation of capsid antigen. This is equivalent to 66.2%. For women over 25 years of age with no capsid antigen detected, remissions were seen only in 25%, but 75% progressed. On the other hand, the remission frequency was 22.2% for women over 30 years old. 77.8% showed a progression decision.

Claims (16)

In a method of diagnosing a human papillomavirus (carcinoma caused by or related to human papilloma virus) or its pre-stage using human samples, the capsid protein of human papillomavirus (HPV) is expressed. A method as described above, characterized in that it is checked whether or not. 2. The method according to claim 1, wherein the capsid protein is capsid protein L1 or L2. The method according to claim 1 or 2, wherein the carcinoma is anogenital cancer. 4. The method of claim 3, wherein the anogenital cancer is cervical cancer. The method according to any one of claims 1 to 4, wherein the human body sample is a smear or a biopsy sample. 6. A method according to any one of claims 1 to 5, wherein expression is determined at the nucleic acid level. 7. The method of claim 6, wherein it is determined whether there is an mRNA encoding the human papillomavirus capsid protein or another replication factor specific for the capsid protein. 7. The method of claim 6, wherein it is determined whether there is a serum response and / or immune response to the capsid antigen. 6. The method according to any one of claims 1 to 5, wherein the determination of expression is performed at the protein level. 10. The method according to any one of claims 1 to 9, wherein the determination of expression is performed at the nucleic acid level and at the protein level. The method according to claim 9 or 10, wherein the determination of the expression of the envelope protein is performed using an antibody directed to the capsid protein. In a test kit of human samples caused by human papillomavirus or involving human papillomavirus (HPV), or a pre-stage thereof, the test kit can be used to determine whether the HPV capsid protein is expressed. The above-described test kit, comprising: 13. The test kit according to claim 12, wherein the reagent comprises a monoclonal antibody directed to HPV capsid protein, in particular directed to capsid protein L1 or L2. 14. The test kit according to claim 13, wherein the antibody is immobilized on a solid support. 13. The test kit according to claim 12, comprising an anti-mouse immunoglobulin in combination with an enzyme, preferably a peroxidase, as a test reagent. 13. The test kit according to claim 12, which is suitable for performing an ELISA test.