WO2008075790A2 - Méthode de détection de cancers à l'aide d'acides nucléiques présents dans le sang, - Google Patents

Méthode de détection de cancers à l'aide d'acides nucléiques présents dans le sang, Download PDF

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WO2008075790A2
WO2008075790A2 PCT/JP2007/075053 JP2007075053W WO2008075790A2 WO 2008075790 A2 WO2008075790 A2 WO 2008075790A2 JP 2007075053 W JP2007075053 W JP 2007075053W WO 2008075790 A2 WO2008075790 A2 WO 2008075790A2
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nucleic acids
detecting
cancer
cancer according
blood sample
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PCT/JP2007/075053
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WO2008075790A3 (fr
WO2008075790A8 (fr
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Tomoko Okasaki
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Olympus Corporation
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C12Q1/6886—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6806—Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/156—Polymorphic or mutational markers

Definitions

  • the present invention relates to a method of detecting cancer using nucleic acids obtained from a blood sample.
  • the nucleic acids are prepared by removing nucleic acids originated from normal cells and by retrieving nucleic acids originated from tumor cells.
  • Non-Patent Document 1 Detecting cancer by using a blood sample is preferable because when compared with endoscopic examination or biopsy, it is less invasive and reduces the burden on patients.
  • a recent study clarified that plasma DNA or serum DNA in circulating blood increases in patients with diseases such as lung cancer (for example, Non-Patent Document 1).
  • a method for detecting the presence or absence of a medical condition in an individual's tissue, cell types and organs includes the following steps of: a) retrieving a bodily fluid such as by collecting blood from the individual; b) extracting total-free floating DNA contained in the sample, and by detecting the amount of the total free DNA or by identifying DNA methylation patterns determining the tissue, cell types or organs in which the free floating DNA originated; c) determining the presence or absence of a medical condition based on the presence or total amount of free floating DNA (i.e. Patent document 1).
  • serum or plasma in blood in which other cell components have been removed are mainly used as the body fluid sample, when detecting a cancer using the free floating DNA in blood sample.
  • Trace amounts of nucleic acids originated from tumor cells are contained in blood; hence it is preferable to remove nucleic acids originated from normal cells as much as possible.
  • cancer is detected from free floating total DNA contained in the sample, thus it is preferable to use a sample in which normal cells have been removed in advance, prior to retrieving the free floating DNA.
  • Methods of separating plasma and serum specimen from blood samples include methods such as manually extracting plasma fraction, and using a blood collection centrifugation tube (blood collection tube) containing polymeric gel for isolation of specimen supplied by Becton Dickinson and Company (BD Vacutainer PPT Plasma Preparation Tube, Reorder Number: 362788).
  • a blood collection centrifugation tube blood collection tube
  • polymeric gel for isolation of specimen supplied by Becton Dickinson and Company (BD Vacutainer PPT Plasma Preparation Tube, Reorder Number: 362788).
  • Patent document 2 a method of screening patients for cancer or precancerous stage; the method also uses samples which are tissue or body fluid sample containing exfoliated cells or cellular debris.
  • This method includes detection of nucleic acids in which the length is longer than 200 base pairs (hereinafter abbreviated as 'bp'), and the presence of the fragments indicates a positive result for presence of cancer or precancerous growth.
  • nucleic acids extracted from sample in which the length of the fragments is less than approximately 200 bp are fractionated and removed as nucleic acids originated from normal cells, so as to screen for cancer or precancerous growth using only the remaining nucleic acids in which the fragment lengths are longer than approximately 200 bp.
  • Patent document 2 supported the apoptosis theory and proposed that nucleic acids originating from normal cells are cleaved into fragments shorter than approximately 200 bp in length. Therefore, the inventers believed that the nucleic acid fragments longer than approximately 200 bp are derived from tumor cells.
  • Non-Patent Document 1 Fournie et al. (1995) Cancer Letters 91 (2): p221 - 227
  • Patent Document 1 Japanese Unexamined Patent Application Publication
  • Patent Document 2 Japanese Unexamined Patent Application Publication
  • nucleic acids originated from normal cells and nucleic acids originated from tumor cells has not yet been established, it is presumed that there are still large amounts of nucleic acids originated from normal cells contained in the prepared nucleic acids sample by the method (2), which increases background noise upon detection of a target nucleic acid.
  • genetic change refers to any types of DNA alterations including such as base change, deletion, duplication, amplification, polymorphism, microsatellite instability, LOH, epigenetic modification.
  • nucleic acids originated from normal cells in particular, for those retrieved from normal leukocytes are mainly 10,000 to 20,000 bp in lengths, thus it has been difficult to detect cancer using nucleic acids of a fragment length longer than 200 bp in the method (2) described above, due to large amounts of contamination of nucleic acids originated from normal leukocytes.
  • the present invention was conceived in view of the above-described circumstances and has as its objective the provision of a method of cancer detection with a high level of sensitivity and precision by reducing background noise upon detecting nucleic acids originated from tumor cells. This is achieved after the subsequent treatments of subjecting nucleic acids in blood samples to fractionation, followed by removal of higher molecular weight nucleic acids, before lower molecular weight nucleic acids are retrieved.
  • the present invention is a method for detecting cancer using nucleic acids in blood samples, the method including the following steps: (a) removing nucleic acids originated from normal cells and retrieving nucleic acids originated from tumor cells from nucleic acids extracted from blood samples, and (b) detection of cancer from the nucleic acids obtained in the step (a).
  • the present invention is a provision of cancer detection method in which the blood sample may be plasma or serum.
  • the present invention is a provision of cancer detection method in which the nucleic acid may be DNA. Furthermore, the present invention is a provision of cancer detection method in which the normal cells may be normal leukocytes.
  • the present invention is a provision of cancer detection method in which the step (a) may include a removal of nucleic acids of a fragment length longer or equal to 5,000 base pairs from nucleic acids extracted from the blood sample.
  • the present invention is a provision of cancer detection method in which the step (a) may include a removal of nucleic acids of a fragment length longer or equal to 500 base pairs from nucleic acids extracted from the blood sample.
  • the present invention is a provision of cancer detection method in which the step (a) may include a retrieval of nucleic acids of a fragment length shorter or equal to 450 base pairs from nucleic acids extracted from the blood sample.
  • the present invention is a provision of cancer detection method in which the step (a) may include a retrieval of nucleic acids of a fragment length shorter or equal to 200 base pairs from nucleic acids extracted from the blood sample.
  • the present invention is a provision of cancer detection method in which the step (a) may include a removal of nucleic acids of a fragment length longer or equal to 10,000 bases from nucleic acids extracted from the blood sample.
  • the present invention is a provision of cancer detection method in which the step (a) may include a removal of nucleic acids of a fragment length longer or equal to 1,000 bases from nucleic acids extracted from the blood sample.
  • the present invention is a provision of cancer detection method in which the step (a) may include a retrieval of nucleic acids of a fragment length shorter or equal to 900 bases from nucleic acids extracted from the blood sample.
  • the present invention is a provision of cancer detection method in which the step (a) may include a retrieval of nucleic acids of a fragment length shorter or equal to 450 bases from nucleic acids extracted from the blood sample.
  • the cancer detection method of the present invention is further characterized in that the step to remove nucleic acids originated from normal cells and to retrieve nucleic acids originated from tumor cells may be performed by employing any single or combination of the separation techniques of filtration, electrophoresis or centrifugation.
  • the filtration technique employed in the cancer detection method of the present invention may be performed by ultrafiltration method.
  • the ultrafiltration techniques employed in the cancer detection method of the present invention may use an ultrafiltration membrane in which the Molecular Weight Cut Off (MWCO) is 50 to 1,000 kD.
  • MWCO Molecular Weight Cut Off
  • the centrifugation technique employed in the cancer detection method of the present invention may be performed by ultracentrifugation.
  • the cancer detection step in the cancer detection method of the present invention may include analyzing the presence or absence of generic changes in the target nucleic acids sequences for the cancer diagnosis.
  • the target region of base sequence containing cancer markers referred to in the cancer detection method of the present invention may include microsatellite sequences.
  • the generic change referred to in the cancer detection method of the present invention may be loss of hererozygosity.
  • the present invention is a provision of cancer detection method may include a step of amplifying nucleic acids obtained in the step (a).
  • the amplification step employed in the cancer detection method of the present invention may be performed by polymerase chain reaction (PCR) method.
  • PCR polymerase chain reaction
  • the amplification step employed in the cancer detection method of the present invention may be performed by real-time PCR method.
  • the cancer detected in the cancer detection method of the present invention may be lung cancer.
  • the present invention enables detection of trace amounts of nucleic acids sometimes present in a whole blood sample, that are derived from tumor cells, with a high level of sensitivity and precision. This is achieved by effectively removing nucleic acids originated from normal cells, in particularly from normal leukocytes, and retrieving only nucleic acids originated from tumor cells.
  • cancer can be detected at early stage and the medical condition can be diagnosed with a high degree of precision by obtaining a small amount of whole blood sample from patients.
  • FIG. 1 shows a flow chart of the preferred cancer detection method according to the present invention.
  • FIG. 2 is a schematic representation of a step of fractionation of plasma from a blood sample by centrifugation.
  • FIG. 3 is a gel photograph showing nucleic acids band pattern results obtained from nucleic acids isolated from plasma which is fractionated by an ultrafiltration membrane YM-50. Each fraction of nucleic acids is subjected to electrophoresis by agarose gel followed by staining with ethidium bromide.
  • the arrow A indicates bands with approximately 10,000 to 20,000 base pairs in length, and the arrow B indicates bands with approximately 180 base pairs in length.
  • 'Blood sample' herein in the present invention refers to blood or prepared samples from blood. This includes, however is not limited to, whole blood from sampled blood, samples such as plasma or serum which are experimentally fractionated from the whole blood, and samples in which chemicals are added thereinto. Samples such as serum or plasma are preferred.
  • the blood sample herein refers to clinically obtained specimen from individuals including healthy individuals, patients with disease, suspected patients with disease, or patients with a special physiological condition, such as pregnancy, which requires clinical attention.
  • samples are not limited to human sources. Samples may include materials from other animal sources, such as from mouse or monkey.
  • 'Plasma' herein refers to a supernate (liquid fraction) obtained from centrifugation of whole blood treated with anticoagulant by methods know in the art.
  • the anticoagulant can be any substances which prevent blood from coagulating. This includes, for example, EDTA (ethylenediamine tetraacetic acid), sodium citrate, and heparin.
  • 'Serum' herein refers to a supernate obtained from centrifugation of whole blood without the anticoagulant treatment by methods known in the art.
  • a glass tube treated with silicon when blood is sampled from patients and other biological sources. This is because the silicon treatment prevents nucleic acids in the blood sample from attaching to the wall of the tube. Furthermore, it is preferable to sample blood with a tube containing a density separating medium such as in a special gel formulation in order to obtain plasma or serum.
  • 'Blood samples' herein refers in the present invention includes thawed blood samples after freeze preservation, as well as fresh blood immediately after being collected from the source. It is preferable that the frozen blood sample is maintained under a suitable environment of -20 to -80 0 C until thawing and processing.
  • Nucleic acids' herein refer to deoxyribonucleotide (DNA) or ribonucleotide (RNA). This also includes nucleotide analogues which can function as natural nucleotides in the same manner.
  • the nucleic acids are preferred to be DNA since a genetic change occurring in tumor cells can be directly detected.
  • nucleic acids are extracted or isolated from a blood sample, and nucleic acids originated from tumor cells are retrieved after removing nucleic acids originated from normal cells.
  • nucleic acids originated from normal cells which are present in large quantities in blood sample By removing nucleic acids originated from normal cells which are present in large quantities in blood sample, and retrieving nucleic acids originated from tumor cells, the proportion of nucleic acids originated from tumor cells in a whole sample used in the detection is increased in order to improve the sensitivity and precision of nucleic acids detection and analysis.
  • 'Normal cell' herein refers to a non-cancerous cell.
  • 'Tumor cell' refers to a cell undergoing abnormal cell differentiation and proliferation, which includes a malignant neoplastic cells (cancerous cells) as well as premalignant preneoplastic cells (precancerous cells).
  • erythrocytes red blood cell
  • leukocytes white blood cell
  • thrombocytes platelets
  • Nucleic acids originated from normal cells contained in a blood sample are often in longer fragment lengths. This may be because most of the cells contained in the whole blood are alive just prior to the blood being collected, so as to minimize exposure of the nucleic acids to nuclease degradation. Therefore less DNA cleaved into smaller fragments are contained in the sample.
  • nucleic acids originated from normal cells and nucleic acids originated from tumor cells immediately after necrosis has occurred simply from their size, when blood sample is used for the detection.
  • nucleic acids originated from both normal cells and from tumor cells will be contained in both fractions when nucleic acids in a blood sample are fractionated into nucleic acids with high molecular weight and low molecular weight.
  • nucleic acids derived from tumor cells are expected to be shorter in fragment length.
  • nucleic acids when nucleic acids are fractionated into nucleic acids with high molecular weight and low molecular weight, the proportion of content of nucleic acids originated from tumor cells is higher in the fraction with low molecular weight nucleic acids in which most of nucleic acids originated from normal cells are removed, compared to the fraction with high molecular weight nucleic acids rich in nucleic acids originated from normal cells.
  • nucleic acids originated from normal cells are nucleic acids with higher molecular weight which are not cleaved into shorter fragments, by fractionating and removing nucleic acids with higher molecular weight extracted or isolated from a blood sample, removal of the nucleic acids originated from normal cells, which become background noise upon detection of nucleic acids, can be achieved.
  • nucleic acid fraction in which higher molecular weight thereof are removed, trace amounts of nucleic acids originated from tumor cells in the blood sample can be detected with a high level of sensitivity and precision.
  • the technique to remove nucleic acids originated from normal cells and to retrieve nucleic acids originated from tumor cells in blood sample are not limited thereto. Any techniques may be employed which enable differentiation of mixtures of nucleic acids into different sizes.
  • techniques include filtration, electrophoresis and centrifugation, and the techniques can be combined thereof.
  • fraction by filtration may be preferably employed due to its simplicity. Fractionation by filtration is performed with filters such as ultrafiltration membranes and filters with different pore sizes.
  • nucleic acids with higher molecular weight originated from normal cells are retained on the surface of the membrane, whereas nucleic acids with lower molecular weight pass through the membranes and are contained in a filtrate.
  • removal of only nucleic acids with high molecular weight is achieved by recovering nucleic acids with low molecular weight in a filtrate from a mixture of nucleic acids with different sizes by employing the ultrafiltration membranes.
  • the size of nucleic acids retained by a filter with molecular weight cut off (MWCO) of 50 kD (YM-50) on its surface is 240 to 475 base pairs in length for double-stranded nucleic acid, and 475 to 950 base lengths for single-stranded nucleic acids.
  • Size of nucleic acids retained by a filter in which the MWCO is 100 kD (YM-100) is 475 to 1,450 base pairs in length for double-stranded nucleic acids, and 950 to 2,900 base lengths for single-stranded nucleic acids.
  • Size of nucleic acids retained by a filter in which the MWCO is 1,000 kD is 4,800 to 5,700 base pairs in length for double-stranded nucleic acids, and 9,500 to 12,000 base lengths for single-stranded nucleic acids.
  • the YM-100 when employed for filtration, most of the nucleic acids in which the length is shorter than or equal to 450 base pairs in length or 900 base lengths are collected in the filtrate.
  • nucleic acids in which the length is shorter than or equal to 6,000 base pairs in length or 12,000 base lengths are collected in the filtrate.
  • a size of nucleic acids retrieved from normal leukocytes is mainly 10,000 to 20,000 base pairs in length. Therefore, filtration with an ultrafiltration membrane is preferably conducted by employing a membrane in which the MWCO is between 50 and 1,000 kD, and more preferably employing a ultrafiltration membrane in which MWCO is between 50 and 100 kD. Furthermore, the most preferable MWCO size of the ultrafiltration membrane is 50 kD.
  • ultracentrifugal separation may be preferably employed. This is because enough centrifugal force required for fractionating nucleic acids with high molecular weight and low molecular weight can be obtained. Nucleic acids with high molecular weight and low molecular weight can also be fractionated by density gradient centrifugation of under between 70,000 and 100,000 xg- Furthermore, nucleic acids with low molecular weight can also be obtained with more concentrated status by the density gradient centrifugation due to the ultracentrifugation force.
  • nucleic acids may be extracted or isolated from a blood sample prior to performing the step (a) for obtaining nucleic acids in the blood sample.
  • 'Extraction or isolation of nucleic acid' herein refers to collect nucleic acids while as much of most of the other components contained in a blood sample as possible are being removed.
  • the other components include proteins, lipids and etc.
  • proteins proteins, lipids and etc.
  • background noise will increase upon detection of cancer.
  • nucleic acids free of proteins, lipids and other cellular components in the present invention.
  • Nucleic acid extraction or isolation methods used herein are not limited thereto; any extraction or isolation by any of the various techniques known in the art can be employed.
  • nucleic acids may be extracted from blood samples by methods employing such materials as glass beads, silica particles, or diatomite; chemical extraction methods employing chemicals such as guanidine thiocyanate, phenolic acid or chloroform; or other methods such as those employing gelatin gel (For example, US Patent No. 6,329,179, or International Publication No. WO 01/42504).
  • Nucleic acid may also be extracted by commercially available nucleic acid extraction kits such as the 'QIAamp Blood Kit' (QIAGEN) used according to the manufacture's instructions.
  • QIAGEN 'QIAamp Blood Kit'
  • Extracted nucleic acids may be further purified by employing chromatography techniques such as size exclusion chromatography or ion exchange chromatography.
  • step (b) of the present invention cancer is detected using nucleic acid samples obtained from the step (a).
  • the detection method is not limited thereto as long as cancer is detected from nucleic acids extracted or isolated from the step (a).
  • the methods include determination of the quantity of nucleic acids and analyzing the presence or absence of genetic changes in base sequences in which known cancer makers are present.
  • the method for analyzing the presence or absence of generic changes in nucleic acids sequences of specific marker genes involved in certain types of cancer is more preferable since influence by individual variation in each sample is small.
  • the region of a base sequence where a cancer marker is present is not limited thereto; it includes any base sequence regions where any modifications, which are derived from tumor cells are detected when the sequence is compared with those derived from normal cells.
  • the base sequence regions include specific marker genes involved in a certain types of cancer, cancer suppressor genes, any cording regions in nucleic acid sequences which may relates to cancer, and regions including microsatellite sequences.
  • Microsatellite is a region found in DNA which contains short (2 - 7) base length sequences repeated from two to a couple of dozens times. Microsatellite instability (which changes the length of the repeated sequence) at certain loci is often observed in DNA samples from tumor tissues. Therefore it is preferable that the base sequence region in which microsatellites are contained is selected as a cancer marker.
  • base sequences are not limited according to the present invention to those base sequences in which known cancer markers are present, and can includes any genetic mutations detected in genes derived from a tumor tissue.
  • base sequences mutation of genes involves in cancer includes deletion mutation, loss of heterozygosity (LOH) and DNA methylation.
  • LOH is a deletion or other mutational event within the normal allele that renders the cell either hemizygous (one deleterious allele and one deleted allele) or homozygous for the deleterious allele. Since LOH is observed in chromosomes of most of tumor tissues, it is preferable to detect a presence or absence of LOH.
  • Method of detecting a presence or absence of genetic changes of the target nucleic acid sequences of specific cancer marker genes is not limited thereto; it includes any methods which can detect and analyze regions of base sequence.
  • the detection methods include: size fractionation, identification of base sequence, and detection of a specific base sequence (hereinafter, called a target sequence).
  • the size fractionation can be performed by such as gel electrophoresis.
  • Determination of nucleic acid sequences can be performed by a variety of methods such as commercially available DNA sequencers.
  • Detection of a target sequence can be performed by techniques such as hybridization.
  • Hybridization method is performed by employing an oligonucleotide probe including a complementary sequence of the target so as to hybridize with the target sequence in a sample. Hence the presence or absence of the target sequence in the sample can be detected.
  • the probe is labeled with a substrate such as nylon or magnetic beads to detect only the target nucleic acid sequence in a sample, which hybridizes with the probe.
  • the probe In order to detect trace amounts of nucleic acids, it is preferable to label the probe with radioactive isotopes or florescence substances.
  • mass spectrographic analysis methods known in the art used in the detection of trace amounts of substance such as capillary electrophoresis, high performance liquid chromatography (HPLC) 5 thin-layer chromatography (TLC), and hyper diffusion chromatography may also be employed.
  • HPLC high performance liquid chromatography
  • TLC thin-layer chromatography
  • hyper diffusion chromatography may also be employed.
  • a modified probe supplied by Chiron Diagnostics
  • Chiron Diagnostics which enables to hybridize multiple probes labeled with a florescence substance with a target sequence, allows amplification of the florescence signals thereby increasing the detection sensitivity.
  • the step (b) contains a step to amplify nucleic acids obtained in the step (a) in order to increase the sensitivity of detection.
  • Amplification techniques of nucleic acids employed in the present invention are not limited thereto, as long as the amplification of a target sequence region is achieved.
  • PCR polymerase chain reaction
  • Q beta replicase amplification amplification method
  • transcriptional amplification amplification method for amplifying target nucleic acid sequences.
  • PCR or real-time PCR methods may be preferably selected as the amplification method.
  • the real-time PCR method is more preferably employed since quantification analysis can also be performed.
  • Other techniques such as cycle-elongation (single strand amplification) may also be combined to normal PCR cycles.
  • a target sequence may be amplified from nucleic acids obtained in the step (a) as a template, and using a pair of primers designed to amplify a region containing a target sequence of a cancer specific gene.
  • the amplified nucleic acid fragments are fractionated by electrophoresis employed with materials such as agarose-gel or polyacrylamide gel, and stained -with a fluorescent dyes such as ethidium bromide or SYBR Green® (Molecular Probe Inc.).
  • the resultant patterns of bands are compared with nucleic acids retrieved from a blood sample free of tumor cells as a positive control. Patterns with bands of different sizes or with additional bands when compared with the positive control indicate the presence of bands related to the disease.
  • cancer can be detected from the presence or absence of band patterns different from the positive control. Furthermore, cancer can be detected and analyzed more specifically by identifying base sequences of the bands which generated the different patterns, from the positive control.
  • Types of cancer which can be detected according to the invention are not limited thereto.
  • lung cancer pancreas cancer, liver cancer, prostate cancer, and gastric cancer may be included.
  • lung cancer is preferred since a distinctive amplification of a target sequence from the nucleic acids in a blood sample can be expected.
  • Experiment 1 A whole blood sample (5 ml) obtained from a cancer patient 1 was centrifuged at a room temperature at 1,600 xg for 20 minutes and was separated into three layers (fractions) by component (serum, leukocytes and hematocyte). The serum layer (2ml) was transferred into other tube without the leucocyte fraction, which is buffy coat, is being contaminated.
  • nucleic acid solution (I) was obtained from 2 ml of serum, and 400 ⁇ L nucleic acid solution (I) derived from the serum was obtained. Likewise, 400 ⁇ L nucleic acid solution (II) was also derived from 200 ⁇ L of the leukocytes fraction (buffy coat). The extraction method followed the manufacture's instructions in the same manner.
  • a Microcon Centrifugal Filter Unit YM-50 (MILLIPORE) was set up by placing the unit into a vial according to the manufacture's instruction.
  • the nucleic acid solution (I) 400 ⁇ L derived from the serum was transferred into a sample reservoir of the unit.
  • the solution was concentrated by centrifuging the filter unit containing the nuclei acid solution derived from the serum at 14,000 xg for 10 minutes until a volume of the solution (retentate (A)) on the sample reservoir was reduced down to 10 ⁇ L. After the centrifugation, an eluate (A) is eluted through the filter unit and collected into the vial.
  • the eluate (A) was then transferred into a Microcon Centrifugal Filter Unit YM-30 (MILLIPORE) in which the pore size was smaller than that of the YM-50, and centrifuged at 14,000 xg for 20 minutes until the volume of the eluate (A) (retentate (B)) is reduced down to lO ⁇ L.
  • the retentate obtained after the centrifugation is called as retentate (B).
  • the 10 ⁇ L of retentates (A) and (B) were subjected to agarose gel (1%) electrophrosis and the fractionated nucleic acids were stained with ethdium bromide.
  • the nucleic acid solution (II) derived from the leukocytes fraction (buffy coat) was placed on the same agarose gel.
  • Lane 1 is a standard DNA marker ( ⁇ Hind III digest, TaKaRa Biolnc, Japan) in which ⁇ phage DNA is digested with a restriction enzyme, Hind III.
  • Lanes 2 and 3 are the retentate (B);
  • lane 4 is the buffy coat derived nucleic acid solution (II) as the positive control; and
  • lanes 5 and 6 are the retentate (A).
  • Lane 7 is also a standard DNA marker (200 bp DNA Ladder Marker, TaKaRa Biolnc, Japan).
  • an arrow A indicates a band in the region of 10,000 to 20,000 bp
  • an arrow B indicates a band with approximately 180 bp.
  • nucleic acids contained in the retentate (B) of the YM-30 after passing through the YM-50 can be analyzed as mainly consisting of low molecular weight nucleic acids with less than or equal to approximately 180 bp in length.
  • the retentate (A) which was retained on the sample reservoir of the YM-50 that is, nucleic acids solution which did not pass through the YM-50 column, can be analyzed as mainly consisting of high molecular weight nucleic acids of approximately 10,000 to 20,000 bp in length. Furthermore, nucleic acids contained in the solution (II) derived from the leukocytes fraction (buffy coat) appears as approximately the same length as the high molecular nucleic acids which did not pass through the YM-50 column.
  • nucleic acids derived from normal leukocytes can be removed from the nucleic acid sample extracted from serum by employing the TM-50 column.
  • LH Loss of heterozygosity by fragments analysis was performed with the nucleic acid retentates (A) and (B) derived from serum fraction, together with the nucleic acid solution (II) derived from leukocytes in Example 1.
  • the LOH analysis was conducted with a standard method using a microsatellite region with the ID number D3S1293 which is found in transforming growth factor-beta receptor 2 gene on human chromosome 3p22, amplified with a forward primer of the sequence number of 1 (D3S1293 forward primer) and a reverse primer of the sequence number of 2 (D3S1293 reverse primer).
  • D3 S 1293 forward primer was labeled with a fluorescent dye HEX.
  • a sequence number 3 in attached Sequence Listing shows a sequence of the microsatellite D3 S 1293.
  • the D3 S 1293 forward primers and D3 S 1293 reverse primer anneal at the positions of the 47 th to 64 th base sequence and at the 160 th to 178 th base sequence of the nucleic acid of the sequence number 3 which is the microsatellite D3S1293 sequence, respectively.
  • PCR reaction mixes were prepared with the 20 ng or 2 ng of the genomic DNA contained in the retentates (A), (B) or the leukocytes nucleic acid solutions (II) obtained in Example 1.
  • Each genomic DNA was mixed with 20 pmol each of the D3S1293 forward primer and the D3S1293 reverse primer, a dNTP mix to the final concentration of 0.2 mM, and 5 ⁇ L 10 x Ex Taq Buffer (TaKaRa) to make the final volume of 50 ⁇ L with reverse osmosis (RO) water.
  • TaKaRa Ex Taq (1.25 units) (TaKaRa Biolnc, Japan) was added to each PCR reaction mix, and the PCR reaction mixes were subjected to a PCR cycle of 30 seconds at 94 0 C, 30 seconds at 65°C, 30 seconds at 74°C; the cycle was repeated with 25 or 35 times.
  • PCR products of microsatellite D3S1293 sequence were obtained; which were amplified from the genomic DNA contained in the retentate (A), the retentate (B) and the leukocytes derived nucleic acid solution (II), serving as the template nucleic acids in the PCR reaction.
  • Aliquots of the amplified PCR products (1 ⁇ L) were analyzed by a capillary sequencer (Genetic Analyzer 3130x1, Applied Biosystems).
  • Example 2 From the result obtained in Example 2, the LOH fragment analysis was performed with the retentate (B) derived from serum. On the other hand, the analysis was not able to achieve using the retentate (A) derived from serum and the leukocytes (buffy coat) derived nucleic acid solution (II).
  • the retentate (A) containing higher molecular weight nucleic acid which did not pass through the YM-50 was unable to detect cancer due to the large amounts of contamination of nucleic acid derived from normal leukocytes, it is obvious that detection of cancer is possible by analyzing trace amounts of fractionated low molecular weight nucleic acids retrieved from tumor cell, since a large proportion of nucleic acid derived from normal leukocytes have been removed by employing the YM-50.
  • Example 3 LOH analysis was conducted with the retentate (B) derived from serum, as seen in Example 2.
  • detection of cancer from trace amounts of nucleic acids retrieved from tumor cells can also be performed with a low molecular nucleic acid fraction which passed through the YM- 100 column.
  • the cancer detection method in the present invention offers early detection of cancer and to monitor the progression of cancer in patients, thereby allowing specific treatments which suit to individual patients to be performed.

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Abstract

L'invention porte sur une méthode de détection de cancers utilisant les acides nucléiques présents dans des échantillons de sang, et comprenant les étapes suivantes: (a) élimination des acides nucléiques provenant de cellules normales et récupération des acides nucléiques provenant de cellules tumorales dans les échantillons de sang; et détection (b) de cancers à partir des acides nucléiques obtenus en (a). Selon l'invention, un cancer peut être détecté avec haut degré de sensibilité et de précision en utilisant les acides nucléiques à l'état de traces procenant des cellules tumorales et quelquefois présents dans les échantillons de sang.
PCT/JP2007/075053 2006-12-20 2007-12-19 Méthode de détection de cancers à l'aide d'acides nucléiques présents dans le sang, WO2008075790A2 (fr)

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Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1634966A2 (fr) * 1997-05-30 2006-03-15 Xenomics Procédé de détection de séquences d'acides nucléiques dans de l'urine

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1634966A2 (fr) * 1997-05-30 2006-03-15 Xenomics Procédé de détection de séquences d'acides nucléiques dans de l'urine

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
BREMNES R M ET AL: "Circulating tumour-derived DNA and RNA markers in blood: a tool for early detection, diagnostics, and follow-up?" LUNG CANCER, ELSEVIER, AMSTERDAM, NL, vol. 49, no. 1, 1 July 2005 (2005-07-01), pages 1-12, XP004976730 ISSN: 0169-5002 *
JAHR S ET AL: "DNA fragments in the blood plasma of cancer patients: quantitations and evidence for their origin from apoptotic and necrotic cells." CANCER RESEARCH 15 FEB 2001, vol. 61, no. 4, 15 February 2001 (2001-02-15), pages 1659-1665, XP002494286 ISSN: 0008-5472 *
SCHWARZENBACH HEIDI ET AL: "Detection and characterization of circulating microsatellite-DNA in blood of patients with breast cancer." ANNALS OF THE NEW YORK ACADEMY OF SCIENCES JUN 2004, vol. 1022, June 2004 (2004-06), pages 25-32, XP002494292 ISSN: 0077-8923 *
WANG MENGJUN ET AL: "Preferential isolation of fragmented DNA enhances the detection of circulating mutated k-ras DNA." CLINICAL CHEMISTRY JAN 2004, vol. 50, no. 1, January 2004 (2004-01), pages 211-213, XP002494284 ISSN: 0009-9147 *

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