Method of quantifying tumour cells in a body fluid and a...

Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid

Reexamination Certificate

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C435S091200, C435S194000, C536S023100, C536S024300, C536S024330

Reexamination Certificate

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06582904

ABSTRACT:

This application is the National Stage of International Application No. PCT/DE96/02183, filed Nov. 14, 1996. Benefit of priority to 35 U.S.C. §365(b) to German application no. 195 42 795.5, filed Nov. 16, 1995 is claimed herein.
The invention relates to a method for the quantification of tumor cells in a body fluid, in which firstly a reaction is carried out with the sample to be investigated, in which reaction the RNA component of telomerase is specifically amplified, and subsequently the amount of amplified nucleic acid is determined quantitatively, and to test kits suitable therefor.
Virtually all solid malignant tumors have the potential to form metastases. The metastasis process comprises the spread of malignant cells as micrometastases, usually via the blood or lymph to remote organs and the development of autonomous secondary tumors. The extent of metastasis determines the prognosis of an oncosis.
The requirements of tumor prevention or aftercare programs are to diagnose primary tumors or a recurrence or a metastasis early, even before metastases become clinically manifest. This aim cannot yet be satisfactorily met with the available instrumental techniques; in particular, there is still a diagnostic gray zone between circulating tumor cells and incipient formation of metastases in organs. Early diagnosis of circulating malignant cells, for example in peripheral blood of a patient undergoing tumor aftercare would make it possible to apply immunomodulating therapy or polychemotherapy, which is possibly curative, at an early date, that is to say even before organ metastasis becomes manifest. Quantification of the metastases in peripheral blood before and after the therapy represents an important control in such cases.
GB 2 260 811 proposes, for example, a diagnostic method for detecting malignant tumors which are associated with normal cells of a particular body tissue, where the normal cells form at least one gene product specific for this tissue. In this detection method, body fluid, for example blood, in which the cells do not normally occur in a healthy person, is taken from the patient, and the mRNA of the specific gene product is amplified and detected. An example mentioned is tyrosinase for detecting melanoma cells in peripheral blood. However, the disadvantage of this method is that it is linked to tissue-specific gene products, does not allow quantification of the melanoma cells and, moreover, gives false-positive results.
Kim et al. describes the results of an assay with which it was possible to determine telomerase activities in tumor tissues [Kim et al. (1994). Science 266: 2011]. The telomerase activity was detected with a sensitivity of about 1 immortal cell/104 normal cells in 98 of 100 cancer cell cultures and 90 of 101 malignant tumors, and in germinal tissues, but not in 22 normal somatic cell-cultures.
Telomerase is a newly described ribonucleo-protein with reverse transcriptase activity [Shippen-Lentz et al. (1990), Science 247: 546] which uses an integral RNA sequence as template for independent DNA synthesis [Greider et al. (1989). Nature 337: 331] by which new telomeric DNA are synthesized at the ends of the chromosomes. Telomeres consist of highly conserved (TTAGGG)n in tandem sequences with a length of about 5-15 kilobases (kb)/cell genome and have the task of stabilizing the chromosomes on the nuclear membrane and protect the coding genomic DNA from uncontrolled recombination and degradation [Mehle et al. (1994). Cancer Res 54: 236]. Whereas a dynamic equilibrium between shortening of the chromosome ends and de novo synthesis of telomeric sequences by telomerase is postulated in lower eukaryotes, normal human somatic cells show low or undetectable telomerase activity. In addition, telomerase is not growth-regulated, in contrast to other DNA enzymes, since none of the actively proliferating cell cultures showed detectable telomerase activity. Only germ cells and almost all tumor cell lines [Ohyashiki et al. (1994). Cancer Genet Cytogenet 78:64; Rogalla et al. (1994). Cancer Genet Cytogenet 77: 19; Schwartz et al. (1995). Cancer 75: 1094] and tumor tissues (Lunge, [Hiyama et al. (1995). Oncogene 10: 937; Shirotani et al. (1994). Lung Cancer 11: 29], kidneys [Mehle et al. (1994). Cancer Res 54: 236], ovaries [Chadeneau et al. (1995). Cancer Res 55: 2533] and blood [Counter et al. (1995). Blood 85: 2315]) show measurable telomerase activity and a constant telomere length which is retained throughout an infinite number of cell divisions. Activation of telomerase with the stabilization, associated therewith, of the telomere length can therefore be regarded as a critical step in the direction of immortalization of somatic cells.
Feng et al. were able to clone the integral RNA sequence of human telomerase (hTR), which is encoded on the distal segment (q) of chromosome 3. The authors were able to demonstrate, by competitive polymerase chain reaction (PCR), a significant increase in telomerase expression in tumor tissues and in germinal tissues by comparison with normal somatic cells [Feng et al. (1995), Science 269: 1236]. An antisense construct of the hTR sequence caused cell death (apoptosis) in transfected HeLa cells. These data demonstrate stringent repression of telomerase in somatic tissues, as well as the fact that malignant growth depends on the presence of immortal cells and on the activation of telomerase.
The object of the present invention was therefore to develop a method with which it is possible to determine tumor cells quantitatively in a body fluid.
The invention therefore relates to a method for the quantification of tumor cells in a body fluid, in which firstly a reaction is carried out with the sample to be investigated, in which reaction the RNA component of telomerase is specifically amplified, and subsequently the amount of amplified nucleic acid is determined quantitatively, and to test kits suitable therefor. Body fluid means for the purpose of the present invention, for example, blood, urine or else stool, exudates or transudates from body cavities, especially peripheral blood.
Peripheral blood is, for example, taken from the subject by puncturing an artery, vein or finger pad and is transferred into an RNA lysis buffer which comprises, for example, urea or, preferably, guanidinium isothiocyanate, in order to denature any RNases present and to release the nucleic acids from the cells [see, for example, Chomczynski et al. (1987) Anal. Biochem. 162, 156]. The nucleic acids can be isolated from the strongly saline medium of the RNA lysis buffer, for example, by means of silica particles to which all nucleic acids are able to bind [Boom et al. (1990) J. Clin. Microbiol., 29, 495]. The particles are then washed several times with suitable buffer and the bound nucleic acids are eluted. It is subsequently advantageous to hydrolyze any genomic DNA present in the sample using RNase-free DNase in a suitable buffer, so that no false-positive results or excessive background noise result due to false amplification signals, because DNA is possibly still present, in the later amplification of the RNA components of telomerase. This is generally followed by inactivation of the DNase, for example by phenol extraction and/or heat denaturation. It is possible and advantageous, before or, preferably, after treatment of the sample with DNase, also to purify the RNA present in the sample further, for example by chromatographic methods such as ion exchange chromatography, preferably on silica gel.
To check whether possibly interfering genomic DNA is still present in the sample, it is subsequently possible to carry out an amplification reaction with the telomerase-specific oligonucleotide primers which are described hereinafter, in which case the RNA present in the sample is not transcribed to cDNA by a reverse transcription reaction beforehand. Only in the case where the sample is free of telomerase-specific DNA does no amplification

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