Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
2000-08-04
2004-11-23
Fredman, Jeffrey (Department: 1634)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C435S091100, C435S091200, C536S023100, C536S024300
Reexamination Certificate
active
06821726
ABSTRACT:
The invention relates to a method for the quantification of tumor cells in a body fluid, in which firstly the sample to be examined is subjected to a process for the enrichment or depletion of tumor cells and a reaction is carried out on the enriched or depleted tumor cells in which the mRNA encoding the catalytic subunit of the 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 the detection of 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 introduce 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 tumor cells in peripheral blood before and after the therapy represents an important control in such cases.
The life span of eukaryotic cells is, according to the telomer hypothesis, determined by the length of the termini of the chromosomal DNA, the telomers. Thus, according to this theory, telomers have the function of a mitotic clock. Because of the replication mechanism of the DNA polymerases, the telomers are shortened after each cell division by the length of the replication primer. As a consequence, after each cell division, the chromosomes are shorter, reaching a critical length after a certain number of cell divisions. The cells then proceed into a senescent phase where they no longer divide and finally die. However, in some cases this regulation mechanism can be bypassed by a ribonuclearprotein, the telomerase, and the cells become immortal. The telomerase synthesizes, at the 5′-end of the chromosomes, the telomer sequences lost during replication, where the RNA component of the protein (human Telomerase RNA component, hTR) serves as matrix and part of the protein component forms the catalytic subunit (human Telomerase Reverse Transcriptase, hTRT).
In humans, the cells with active telomerase and unlimited life expectancy are, in particular, the germ cells and the hemapoietic stem cells, which are capable of dividing during the entire life span of a person. In addition, increased telomerase activities are also found on activated human B and T lymphocytes. Besides this normal physiological role of telomerase, an increased telomerase activity is found in about 90-95% of all human tumor tissues. The telomerase activity of tumor cells can therefore form the basis for a detection system for disseminated circulating tumor cells in body fluids, which may potentially give rise to metastases.
Telomerase is a ribonucleoprotein with reverse transcriptase activity [Shippen-Lentz et al. (1990), Science 247: 546&pgr; 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.
In 1997, Nakamura et al. characterized a protein component of the catalytic subunit of human telomerase. In comparison with the RNA component of human telomerase (hTR), the mRNA encoding the catalytic subunit of human telomerase (hTRT) correlates considerably better with the telomerase activity (Nakamura T M, Morin G B, Chapman K B, Weinrich S L, Andrews W H, Lingner J, Harley C B, Cech T R (1997): Telomerase catalytic subunit homologs from fission yeast and human. Science 277: 955-9) and is therefore more suitable for cancer diagnosis. Meyerson et al. localized hTRT on the human chromosome 5p and confirmed the strong correlation of the hTRT mRNA detection with the enzymatic activity of human telomerase (Meyerson M, Counter C M, Eaton E N, Ellisen L W, Steiner P, Caddle S D, ziaugra L, Beijersbergen R L, Davidoff M J, Liu Q, Bacchetti S, Haber D A, Weinberg R A (1997): hEST2, the putative human telomerase catalytic subunit gene, is up-regulated in tumor cells and during immortalization. Cell 90: 785-95).
Currently, the standard method for determining the catalytic activity of telomerase is the TRAP assay (Telomeric Repeat Amplification Protocol) [Kim et al. Science (1994) 266: 2011]. Here, the telomerase present in cell extract synthesizes extension products which are subsequently amplified in a polymerase chain reaction (PCR). A densitometric evaluation of the amplification products then permits quantification of the telomerase activity. Depending on the sample material, the test laboratory and the protocol used, the sensitivity of the TRAP assay was stated as being 4-100 cells/batch. Since the crude extract of lyzed cells or tissues is used in the TRAP assay, this method is highly susceptible to interference by inhibitors of the DNA polymerase used in PCR (Taq polymerase).
The TRAP assay is used, for example in WO 98/02581, for the diagnosis of precancerous or cancerous damage to the cervix, endocervix, the vagina or vulva.
WO 97/18322 discloses a method for the quantification of tumor cells in which initially a reaction is carried out with the sample to
Brockmeyer Carsten
Dahm Michael W.
Phelps Robert C.
Fredman Jeffrey
Goldberg Jeanine
Seidman Stephanie L.
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