IMMUNOHISTOCHEMICAL INVESTIGATIONS OF P 16 INK 4 A EXPRESSION IN CARCINOMAS AND HIGH GRADE CERVICAL LESIONS

During the years the role of viruses as a cause of oncological illness was known. One of them is cervical carcinomas, related to Human Papilloma Viruses (HPV’s). There are about 450000 new cases of cervical cancer worldwide each year, and 250000 deaths of this diagnosis. By the diagnosis cervical cancer gets there with two procedures: after mass screening or clinical suspicious for ill. By the availability apparent tumor diagnosis is put with biopsy. By the discreet lesions require utilization modern diagnostic methods. One of them is immunohistochemical method. In situ hybridization (ISH) assays for high-risk human papilloma virus (HR-HPV) and immunohistochemical (IHC) assays for surrogate markers such as p16 INK 4a can be useful in detecting HR-HPV in cervical dysplasia. The aim of the following study was to find difference the level for expression of the tumor specific gene p16 INK 4a in reactive, dysplastic and invasive changes in biopsy specimens from uterine cervix. In the lesions of the uterine cervix, over expression of p16 INK 4a is induced by HPV and is associated with the process of cancerogenesis in the epithelium. Almost 100% of the high grade lesions and the invasive carcinomas demonstrated high level of expression of p16 INK 4a while non dysplastic epithelium is always negative.


INTRADUCTION:
Viruses have important meaning for human pathology.During the years the role of viruses as a cause of oncological illness was known.One of them is cervical carcinomas, related to Human papilloma viruses (HPV's).WHO Regional Office for Europe reported by cervix cancer morbidity.Data for selected countries are presented on fig. 1.There are about 450000 new cases of cervical cancer worldwide each year, and 250000 deaths of this diagnosis.Every year new cases of cervical dysplasia, related to HPV's, are assess at 10 million cases with CIN II/III, 30 million cases with CIN I. Success in diagnostics of this dis-ease is due to the use of Pap-test /citological smear analysis/.However Pap-test gives significant portion of both false-positive and false-negative conclusions.Amendments of the diagnostic procedure are desirable.Aetiological role of papillomaviruses in cervical cancer is established while the role of cellular gene alterations in the course of tumor progression is less clear.Several research groups including us have recently named the protein p16 INK 4a as a possible diagnostic marker of cervical cancer.To evaluate whether the specificity of p16 INK 4a expression in dysplastic and neoplastic cervical epithelium is sufficient for such application we undertook a broader immunochistochemical registration of this protein with a highly specific p16 INK 4a monoclonal antibody.

AIM:
The aim of the following study was to find difference the level for expression of the tumor specific gene p16 INK 4a in reactive, dysplastic and invasive changes in biopsy specimens from uterine cervix.

MATERIALS AND METHODS:
Fifty six biopsy specimens were investigated in the following study.They were separated in four groups as follows: 1 st group -reactive non dysplastic changes (n=13); 2 nd group -different levels of dysplasia to Ca in situ (n=19) 3 rd group -invasive squamous cells carcinoma (n=16) 4 th group -endocervical lesions -Microglandular hyperplasia, Ca in situ and invasive endocervical adenocarcinoma (n=8) Standard 4 µm slides were stained with H&E.An immunohistochemical method with monoclonal antibodies against the tumor-suppressor gene p16 INK 4a was applied on some specimens, according to the instructions provided by the manufacturer (DAKO).The evaluation of the positive immunohistochemical reactions for p16 INK 4a was also semiquantitative (0 when there is no positive reaction, 1+ from 15%to 20% staining of the nuclei and cytoplasm, 2+from 25% to 75% and 3+>75%).Weak cytoplasm staining (<5%) was considered negative.For statistical analysis of the results was used the method of correlative analyses and c2.Values of p<0.05 were considered as statistically significant.

RESULTS:
The results from the expression of p16 INK 4a are presented in table 1.In the cases with dysplastic lesions and invasive carcinomas was found strong correlation between the level of expression of p16 INK 4a and the level of cervical neoplasia (p<0.01).All 13 cases (100%) of non dysplastic cervical lesions are negative for p16 INK 4a (fig.2).The most cases of CIN III group (7cases-87,5%) showed strong (+++) cytoplasmic and nuclear expression of p16 INK 4a in the whole depth of the epithelium (fig.3, 4).Strong mainly nuclear everexpression was found in all invasive cervical adenocarcinomas.(fig.5).

DISCUSSION:
In the lesions of the uterine cervix, overexpression of p16 INK 4a is induced by HPV and is associated with the process of cancerogenesis in the epithelium.These observations show that, the p16 INK 4a immunostaining can make easier the precise identification of displastic cervical epithelial cells on histological specimen.It displays the cellular changes in the infected cell, as a result of the genomic integration of the viral oncogenes.Because of the fact that overexpression is not found in normal, non-transformed cells p16 INK 4a is reliable marker for identification of dysplastic and malignant epithelium.Almost 100% of the high grade lesions and the invasive carcinomas demonstrated high level of expression of p16 INK 4a while non displastic epithelium is always negative.

CONCLUSION:
This statistically significant different expression of p16 INK 4a , in different types of lesions in the uterine cervix, contributes for their identification and makes this marker helpful and objective for the diagnosis of the cervical pathology.So far no indications for a difference in the p16 INK4a staining pattern of cervical lesions caused by different HR-HPV types have been found.The velocity of neoplastic transformation seems to be different for different HR-HPV types, but once the oncogene products of HR-HPV have caused cell cycle disturbances, as one of the first steps in carcinogenesis, overexpression of p16 INK4a should be detectable independent of the individual HR-HPV type.