Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Patent
1998-01-28
2000-02-22
Horlick, Kenneth R.
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
435 5, 935 19, C12Q 168
Patent
active
060278919
DESCRIPTION:
BRIEF SUMMARY
The present invention relates to a method for early detection of HPV-associated carcinomas and extreme dysplasias caused by HPV.
It is known that many people suffer from persisting infections caused by human papilloma viruses (referred to as HPVs below). Furthermore, it is known that over 95% of all anogenital carcinomas, particularly the cervical carcinoma of the uterus, and a considerable percentage of carcinomas in the oropharynx are associated with persisting infections caused by what are called "high-risk types" of the HPVs.
Therefore, in the cancer screening of women smears of the cervix uteri are examined as to changes in the cells, which are caused by persisting infections caused by HPVs. Such changes are divided into differing degrees of severity. If dysplastic cells are detected, the lesion will usually be removed by conization of the cervix uteri. Since this method has been carried out regularly for the early detection of carcinomas, the incidence of invasive cervical carcinoma has decreased considerably.
Nevertheless, the above method includes considerable drawbacks which include, for example: (1) The evaluation of the cell picture is subject to subjective influences which are due to a cytologist and, depending on his experience; may lead to wrong positive or wrong negative results. (2) By the cytologic evaluation it is not possible to make a distinction between lesions which may regress spontaneously and lesions which may change to an invasive carcinoma. Up to 80% of the early lesions regress spontaneously. However, for reasons of safety these lesions are still removed by conization to avoid the risk of a possible malignancy. The conization per se also results in a certain morbidity, i.e. cervical insufficiency, and obstetric complications connected therewith. (3) The cytologic examination is not sensitive enough in some cases, so that in spite of regular cytologic controls, invasive carcinomas are overlooked in some cases.
Recent studies refer to the fact that for the formation of carcinomas and extreme dysplasias, respectively, an uncontrolled expression of HPV genes, particularly genes E6 and E7, is necessary in the case of cells having a persisting infection caused by HPVs. Reference is also made to the fact that in HPV-associated carcinomas, HPV genomes or at least genes E6 and E7 are integrated into the cell DNA and expressed together with cellular sequences.
This could mean that for the uncontrolled expression of the above genes, they have to be expressed together with cellular sequences. Such an expression product could then be suited to indicate at an early stage the formation of HPV-associated carcinomas and extreme dysplasias caused by HPV, respectively.
The applicant investigated the above and found that the addressed expression product stands for the uncontrolled expression of the genes and is thus suitable for the early detection of HPV-associated carcinomas and extreme dysplasias caused by HPV, respectively. It proved to be favorable to detect the expression product in the form of an mRNA having HPV and cellular sequences. A method suited for this purpose comprises the following steps: transcription, primer) and a primer (3' primer) having sequences of the primer of (c), 5' side of the HPV polyadenylation sequence, "nested" primers, and
A body sample in which an mRNA having HPV and cellular sequences may be detected, is taken from a patient. Samples suitable for this purpose are inter alia a smear or surface biopsy, an organ punctate and a biopsy, respectively, blood, sputum, urine, stool, liquor, bile, lymph and a gastrointestinal secretion. A smear or surface biopsy is preferred.
The body sample, preferably a smear or surface biopsy, is taken and prepared as usual. The mRNA is isolated from the body sample according to common methods. It is favorable to use a purchasable extraction kit, e.g. GlassMax, Gibco BRL. Contaminating DNA in the mRNA preparation is removed by common DNase digestion.
The resulting mRNA is subjected to reverse transcription, a common primer being used. T
REFERENCES:
patent: 5821048 (1998-10-01), Howley
Cornelissen, et al., "Uniformity of the Splicing Pattern of the E6/E7 Transcripts in Human Papillomarvirus type 16-Transformed Human Fibroblasts, Human Cervical Premalignant Lesions and Carcinomas", Journal of General Virology--71:1243-1246 (1990).
Emmerich Florian
Von Knebel-Doberitz Magnus
Worner Stefan
Deutsches Krebsforschungszentrum des Offentlichen Rechts
Halluin Albert P.
Horlick Kenneth R.
Smith J. David
Taylor Janell E.
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