Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Patent
1996-06-17
1999-09-14
Myers, Carla J.
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
435 912, 536 2431, 536 2433, C12Q 168, C12P 1934, C07H 2104
Patent
active
059521708
DESCRIPTION:
BRIEF SUMMARY
The present invention is concerned with a method for diagnosing and/or monitoring the evolution of various types of cancers after a chemotherapeutic treatment or after surgery.
It is well known that the diagnosis and the monitoring of the evolution of cancers is carried out, in addition to the observation and the direct examination of the tumors, by the analysis of biopsy samples, or in the case of blood cancers, an examination of the bone marrow. This implies either a surgical intervention, or an invasive operation of the biopsy type or further a bone medulla aspiration using a needle. Actually, in addition to the unpleasant if not dangerous nature of such methods to the patient, it was found that they could furthermore lack accuracy. In the case of certain leukemic diseases for example, the analysis of the sample of bone marrow taken has not made it possible to identify all the malignant clone varieties.
The purpose of this invention is hence to provide a method for diagnosing cancers which, on the one hand, is more accurate and more reliable and which, on the other hand, is easier to carry out without the need of resorting to invasive tests on the patients.
The method for diagnosing and/or monitoring the evolution of cancers, object of the invention and aimed at achieving the above purposes, includes the analysis of the deoxyribonucleic acid (DNA) contained in the blood plasma.
In fact, it has now been possible to demonstrate that patients suffering from different cancer diseases show increased amounts of DNA in the blood plasma. The method of diagnosis according to the invention is hence based on the detection of gene mutations in this plasma DNA, the blood plasma being a human material much more easily accessible that biopsy samples of tumors for example. Thus, oncogene mutations are frequently evidenced in numerous types of malignant tumors and, amongst them the mutations of the ras gene are particularly meaningful. However, the method can be applied to any gene modification of the DNA of the carcinomatous cells, such as mutations and deletions of the genes ras, APC, DCC, P53, etc of any oncogene or anti-oncogene gene (tumor suppressing gene) or furthermore modifications in the microsatellites. It has even been found that different mutations of the ras gene detected in the DNA of the blood plasma could be absent in the DNA of peripheral blood cells or in the case of certain patients suffering from leukemia in the DNA of bone marrow, which tends to confirm the greater reliability of the method according to the invention by comparison to known diagnostic methods.
Generally, the method of diagnosis according to the invention consists in extracting the DNA from blood plasma, in purifying and amplifying this DNA, and thereafter in determining gene mutations or deletions therein, in principle in a comparative test, with the blood plasma of a person presumed to be ill and that of another person in good health, as references.
The scope of the present invention extends to any technique for extracting, purifying or amplifying blood plasma DNA; also, any method appropriate for determining gene mutations can be used.
The diagnostic method according to the invention will now be described in more detail with reference to the two following examples:
EXAMPLE 1
Diagnosis of the cancer of the colon by detection of mutations of the K-ras gene.
In this first application of the method according to the invention, use was made of the determination of mutations in the codon 12 of the K-ras gene contained in the adenocarcinomata of the colon. These mutations occur generally upon transition from stage I adenoma to stage II adenoma, before the deletion or the mutation of gene P53, i. e. at a relatively early stage of the development of the tumor.
Blood samples (20-30 ml) were taken from 15 patients with a colo-rectal adenocarcinoma at different stages of development and heparinized, said patients having received at that time no anticancer drug. Thirteen of the 15 patients underwent subsequently surgery to remove the tumor;
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Anker Philippe
Stroun Maurice
Vasioukhin Valeri
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