WO2013060762A1 - Méthode pour diagnostiquer une maladie basée sur la distribution de l'adn plasmatique - Google Patents

Méthode pour diagnostiquer une maladie basée sur la distribution de l'adn plasmatique Download PDF

Info

Publication number
WO2013060762A1
WO2013060762A1 PCT/EP2012/071117 EP2012071117W WO2013060762A1 WO 2013060762 A1 WO2013060762 A1 WO 2013060762A1 EP 2012071117 W EP2012071117 W EP 2012071117W WO 2013060762 A1 WO2013060762 A1 WO 2013060762A1
Authority
WO
WIPO (PCT)
Prior art keywords
dna
plasma
peak
apoptosis
cancer
Prior art date
Application number
PCT/EP2012/071117
Other languages
English (en)
Inventor
Jochen Geigl
Ellen Heitzer
Eva-Maria Hoffmann
Michael Speicher
Original Assignee
Roche Diagnostics Gmbh
F. Hoffmann-La Roche Ag
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Roche Diagnostics Gmbh, F. Hoffmann-La Roche Ag filed Critical Roche Diagnostics Gmbh
Priority to EP12780167.8A priority Critical patent/EP2771483A1/fr
Publication of WO2013060762A1 publication Critical patent/WO2013060762A1/fr

Links

Classifications

    • 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
    • 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
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/112—Disease subtyping, staging or classification
    • 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 diagnosing a disease, particularly a disease associated with increased apoptosis, and the use of size distribution of plasma DNA in the diagnosis of the disease.
  • Methods of diagnosis usually aim at being minimal invasive, technically robust and universally applicable.
  • Blood analysis has been used as a diagnostic tool for many years, as blood is easily obtainable and the analysis of its components is usually relatively easy and automatable. Additionally, the composition of the blood components usually reflects a variety of diseases.
  • CTC colorectal cancer
  • RT-PCR real-time PCR
  • primers with fluorescence markers are used and the increase of fluorescence depending on the number of cycles is monitored.
  • a disadvantage of this procedure is that it is costly and has limited capability for multiplex high-throughput screenings.
  • our own analyses show that with a low amount of starting material, quantitative PCR does not provide reliable results and that the actual copy numbers are often not determined correctly.
  • BEAMing beads, emulsion, amplification, magnetic
  • the technique comprises four essential steps (Diehl et al, 2005) which is exemplified for a mutation in the APC gene: (i) The total number of plasma DNA fragments comprising APC is determined by real-time PCR; (ii) BEAMing is then used in order to link the amplified plasma DNA to beads, wherein within the beads an emulsion PCR is performed, in order to amplify each individual plasma DNA fragment within a bead to obtain so called “extended beads” which are subsequently broken ("emulsification”); (iii) the mutation status of the extended beads is determined by single base extension (incorporation of fluorochrome-labeled ddNTPS (causing termination of the reaction)) so that plasma fragments with and without mutation differ by the respective fluorochrome; and (iv) the fluorochromes are measured by flow cytometry and the ratio of mutant and wild type alleles
  • the biomarker consists essentially of region- spanning breaks, whose presence and amounts in plasma may be monitored. As translocation break points may vary from tumor to tumor, each primary tumor has to be assessed individually and therefore this approach was referred to as PARE (personalized analysis of rearranged ends) (Leary et al., 2010).
  • CML chronic myeloid leukemia
  • MRD minimal residual disease
  • solid tumors are usually highly heterogenic, which means that the translocation might be present only in a subpopulation of tumors cells. This is a further significant difference to leukemia, as the translocation between chromosomes 9 and 22 is present in all CML cells.
  • a further disadvantage is the high effort for designing biomarkers and the high costs associated therewith.
  • a potential advantage might be that, under suitable conditions, also minimal amounts of tumor might be diagnosed.
  • CTCs constitute as few as 1 cell per lxlO 9 hematologic cells in the blood of patients with metastatic cancer making it difficult to identify and isolate these cells (Pantel et al. 2008). Therefore, cells qualifying as CTCs are at present mainly enumerated without further analyses as they are routinely performed in tissue-based diagnostics.
  • fetal diseases in the maternal blood is of increasing interest.
  • the safety of the procedure as compared to e.g. amniocentesis and chorionic villus sampling is evidently of particular interest as well as with legal standards and moral requirements; further, a comparison to preimplantation diagnostics is evidently of particular interest.
  • With pregnant females it is estimated that 10% to 20% of the plasma DNA of a pregnant female are from the fetus.
  • the development of the so called next- generation sequences methods gave rise to further methods for examining fetal DNA in maternal blood.
  • next-generation plasma sequencing was tested in a multi-center study with 753 pregnant females and showed that, indeed, fetal trisomy 21 was diagnosed in maternal blood with a sensitivity of 100%) and a specificity of 97.9%> (Chiu et al, 201 1).
  • These studies suggest that the analysis of plasma DNA has the potential to substitute traditional prenatal diagnosis.
  • a disadvantage is the high effort needed so far.
  • the methods for the diagnosis of a disease according to the state of the art, particularly for diagnosing cancer or fetal aneuploidy pose technical problems.
  • the object of the present invention to provide new methods for diagnosing a disease or for monitoring therapy. Particularly, it was intended to avoid problems of the methods detailed above.
  • the methods of the present invention are particularly suitable in the predictive or prognostic diagnosis of cancer as well as in monitoring cancer therapy. Additionally, the methods may be employed with pregnant females in order to identify fetal aneuploidy.
  • the object was solved by a method involving the determination of size distribution of plasma DNA, wherein an increased non-apoptosis peak in the size distribution relative to a control is indicative of a disease. As shown in the examples, it could be shown that the size distribution of healthy subjects is different from that of diseased subjects. Exemplary size distributions of plasma DNA are shown in Figures 3 a) and b).
  • Figure 3 a shows a peak around 170 base pairs (bp) referred to "region 1".
  • Figure 3 b) shows the plasma size distribution of a diseased subject.
  • a further peak around 340 base pairs can be identified, which is indicative of the disease.
  • the second peak is referred to as non-apoptotic peak.
  • the present invention provides a method which allows to establish genome-wide copy number changes in relevant genomes, e.g., the genome of the tumor and cancer patients or the genome of the fetus in a pregnant female, from plasma DNA in a cheap, simpler and fast manner.
  • the methods of the present invention are advantageous, because they are easy to carry out and to implement.
  • the obtained data are robust and easy to interpret.
  • the methods are cost- efficient as compared to other methods. They are characterized by high sensitivity and provide information about the complete relevant genome. The methods do not depend on the patient's history, in particular with tumor patients; they do not depend on the knowledge about primary tumors, and are independent from clonality and heterogeneity of the tumor. Finally, significant results are obtained faster than with other methods.
  • the methods are particularly useful to provide information about genomic aberrations such as copy numbers of the genome (especially with cancer patients of the tumor genome and with pregnant females of the genome of the fetus) from the plasma DNA.
  • genomic aberrations such as copy numbers of the genome (especially with cancer patients of the tumor genome and with pregnant females of the genome of the fetus) from the plasma DNA.
  • the DNA is prepared in a manner to allow for easy determination of the mutation status of a selected gene or by next-generation sequencing of the complete genome.
  • data provide the basis in order to conclude how probable it is to detect circulating tumor cells (CTCs) in the blood of a cancer patient.
  • CTCs circulating tumor cells
  • the present invention is particularly useful in the field of oncology.
  • Europe only in 2006, about 3,191,600 new cancer cases were diagnosed and about 1,700,000 events of death could be assigned to tumor diseases. Cancer is still the biggest challenge in the health system.
  • this development is accompanied by the discovery of new molecular genetic bio markers providing essential predictive and prognostic information. Mutations in epidermal growth factor receptor are prominent examples which frequently occur at patients with lung carcinoma and which are usually treated with tyrosine kinase inhibitors such as Gefitinib. However, it is a prerequisite to clarify the mutation status and to monitor the treatment, in order to be able to quickly react on possible resistances developing in the meantime.
  • the method of the present invention has the additional advantage that changes throughout the complete tumor genome can be detected very quickly allowing to diagnose the occurrence of further new clones with other characteristics very fast in "real time”. Accordingly, the present invention will have a great benefit in the field of oncology.
  • the invention is also particularly useful in the field of fetal diagnosis.
  • 2009 there were 70,344 live births in Austria.
  • the age of parturients was 26.4 years on the average, whereas it was 30.0 years in 2009. Due to this demographic development, the need to identify fetal aneuploidy is of increasing relevance.
  • the present invention relates to a method of diagnosing a disease, comprising
  • the present invention relates to a method of diagnosing a disease associated with increased apoptosis, comprising
  • the size distribution is biphasic having a apoptosis peak and a non-apoptosis peak, wherein the apoptosis peak is characterized by a maximum in the range of from 150 bp to 180 bp and wherein the non-apoptosis peak is characterized by a maximum at in the range of from 300 bp to 350 bp,
  • Diagnosing a disease in the context of the present invention has a broad meaning. In the medical field, it is used for a process of attempting to determine and/or identify a possible disease done in order to clarify whether or not a subject is diseased or is going to be diseased. Alternatively or additionally, the extent of the disease may be determined. Accordingly, it may or may not be known whether the subject is suffering or is going to suffer from a disease. This is further exemplified with cancer, but is also true for other diseases. In one alternatively, it could be checked whether a subject is suffering from cancer or is going to suffer from cancer (without showing clinical symptoms yet). The diagnosis is carried out in order to find out whether the subject is healthy or suffering from cancer.
  • the degree and extent of the disease could be determined, too.
  • the method could be used in the monitoring of the health status of a previously or still diseased subject. This could be in the context of the monitoring of a therapy or the recurrence.
  • a disease is an abnormal condition affecting the body of an organism. It is often construed to be a medical condition associated with specific symptoms and signs. It may be caused by external factors, such as infectious disease, or it may be caused by internal dysfunctions, such as autoimmune diseases.
  • disease is often used more broadly to refer to any condition that causes pain, dysfunction, distress, social problems, and/or death to the person afflicted, or similar problems for those in contact with the person. In this broader sense, it includes injuries, disabilities, disorders, syndromes, infections, isolated symptoms, deviant behaviors, and atypical variations of structure and function. In the context of the present invention the terms disease, disorder, morbidity and illness are used interchangeably.
  • Plasma-DNA is intended to relate to DNA present in blood plasma.
  • Plasma which constitutes 55% of blood fluid, is mostly water (92% by volume), and contains dissipated proteins, glucose, mineral ions, hormones, carbon dioxide (plasma being the main medium for excretory product transportation) and importantly for the present invention DNA. Plasma is obtained from blood by removal of cells.
  • plasma-DNA is from a subject's blood sample.
  • subject can mean either a human or non-human animal, preferably vertebrates such as mammals, especially primates, such as humans.
  • the sample is a limited quantity of blood which is intended to be similar to and represent a larger amount of the same.
  • a blood sample may be taken from the subject. Particularly for mammals, this may be conveniently performed by taking venous blood from the subject.
  • Venous blood may be obtained by venipuncture from the mammal, e.g. a patient suspected of having cancer or a pregnant woman, wherein usually only a small sample, e.g.
  • Blood is most commonly obtained from the median cubital vein, on the anterior forearm (the side within the fold of the elbow). This vein lies close to the surface of the skin, and there is not a large nerve supply.
  • Most blood collection in the industrialized countries is done with an evacuated tube system consisting of a plastic hub, a hypodermic needle, and a vacuum tube.
  • blood may also be obtained by any other method known to the skilled person.
  • plasma is isolated from the blood sample.
  • cells may be removed by any suitable method.
  • blood plasma is prepared by spinning a tube of blood usually containing an anti-coagulant in a centrifuge until the (blood) cells precipitate on the bottom of the tube. The plasma is then poured or drawn off. Exemplary method is by centrifugation at 1600g for 10 min and microcentrifugation at 16 OOOg for 10 min.
  • the plasma may be immediately used for analysis or frozen for storage as known to the person skilled in the art.
  • the plasma may be frozen at liquid nitrogen temperatures and stored for long periods of time, being thawed and capable of being reused.
  • blood plasma is isolated as follows: Blood is taken from a subject and collected in container intended for blood collection. Those containers are commercially available and may be used in the method of the present invention. Usually, they comprise an anti-coagulant such as EDTA.
  • An exemplary container is a routine EDTA Vacutainer tubes (BD Biosciences, Heidelberg, Germany).
  • suitable agents known to the skilled person such as formaldehyde (e.g. 4% weight per volume) may be added.
  • a buffer solution adapted for stabilisation at neutral conditions may be present (e.g. 10% neutral buffered solution containing formaldehyde (4% weight per volume)). Blood samples may be gently inverted. Thereafter, the sample may be immediately used or stored until further processing (stored at 4° C and further processed within two hours).
  • centrifugation may encompass several centrifugation steps, after which cells are separated from the liquid phase, in order to improve removal of cells and therefore the quality of the plasma.
  • centrifugation should be rather mild in order to avoid loss of larger DNA molecules.
  • centrifugation may be carried out at about lOOOg to 3000g for several minutes, repeatedly or only once.
  • tubes containing the blood may by centrifuged at 200g for 10 min. e.g. with the brake and acceleration powers set to zero with a subsequent centrifugation step at 1600g for 10 min. The supernatant is collected, transferred to a new tube and spun at 1600g for 10 min. Further details are described in Dhallan et al. (Dhallan et al. 2004, 2007). Thereafter, the obtained plasma can be immediately analyzed or stored (e.g. at -80°C).
  • the method may be carried out by using the QIAamp DNA Blood Mini Kit (#51306, Qiagen, Hilden, Germany) or the Qiagen circulating nucleic acids Kit (Qiagen, Hilden, Germany) according to the manufacturer's instructions.
  • a plasma sample from a subject's blood is provided (see above) and the size distribution of the plasma-DNA is determined.
  • a size distribution is a list of values or a mathematical function that defines the relative amounts of particles present, sorted according to size.
  • the DNA molecules of blood plasma are analyzed for their sizes (e.g. in base pairs (bp) and the amount or number of molecules (DNA or DNA fragments) of the various sizes is determined.
  • the size distribution is determined over a list of size ranges that covers nearly all the sizes present in the plasma.
  • the amount of DNA fragments in each size range is preferably listed in order or presented in a coordinate system, e.g. a two-dimensional space (such as a Euclidian space).
  • the size distribution is shown in a Cartesian coordinate system, specifying each point uniquely in a plane by a pair of numerical coordinates, representing the size of the DNA molecule and the amount of the DNA molecules having the respective size.
  • the size will be shown on the X axis and the amount (or a value reflecting or corresponding to the same) of DNA molecules having this size on the Y axis.
  • it is required to determine the size of the DNA molecules present in the plasma sample. Any suitable method known to the skilled person may be used.
  • Suitable methods include electrophoresis, next-generation DNA sequencing technology, PCR such as real-time PCR, Multiplex Ligation-dependent Probe Amplification (MLPA).
  • MLPA Multiplex Ligation-dependent Probe Amplification
  • DNA electrophoresis is an analytical technique used to separate DNA fragments by size.
  • DNA molecules which are to be analyzed are set upon a viscous medium, the gel, where an electric field induces the DNA to migrate toward the anode, due to the net negative charge of the sugar-phosphate backbone of the DNA chain.
  • the separation of these fragments is accomplished by exploiting the mobilities with which different sized molecules are able to pass through the gel. Longer molecules migrate more slowly because they experience more resistance within the gel.
  • the size of the molecule affects its mobility, smaller fragments end up nearer to the anode than longer ones in a given period.
  • the voltage is removed and the fragmentation gradient is analyzed. For larger separations between similar sized fragments, either the voltage or run time can be increased. Extended runs across a low voltage gel yield the most accurate resolution.
  • the DNA fragments of different lengths may be visualized using a fluorescent dye specific for DNA, such as ethidium bromide, wherein the intensity is reflects the amount of molecules.
  • the gel shows bands corresponding to different DNA molecules populations with different molecular weight. Fragment size determination is typically done by comparison to commercially available DNA markers containing linear DNA fragments of known length.
  • the types of gel most commonly used for DNA electrophoresis are agarose (for relatively long DNA molecules) and polyacrylamide (for high resolution of short DNA molecules). Gels have conventionally been run in a "slab” format, but capillary electrophoresis has become important for applications such as high-throughput DNA sequencing. Electrophoresis techniques used in the assessment of DNA include alkaline gel electrophoresis and pulsed field gel electrophoresis. The measurement and analysis are mostly aided by a specialized gel analysis software. Also next-generation DNA sequencing technology may be employed for size analysis. The high demand for low-cost sequencing has driven the development of high-throughput sequencing technologies that parallelize the sequencing process, producing thousands or millions of sequences at once. Exemplary methods include Massively Parallel Signature Sequencing (MPSS), polony sequencing, 454 pyrosequencing and Illumina (Solexa) sequencing.
  • MPSS Massively Parallel Signature Sequencing
  • polony sequencing polony sequencing
  • 454 pyrosequencing for high resolution
  • primers for different-sized amplicons can be designed. These PCR assays use different reverse and/or forward primers, and one common or different binding probe(s). PCRs may be set up in a reaction tube, followed by amplification in the tube. Amplification data may be collected and analyzed by suitable software, e.g. Sequence Detection System Software (Ver. 1.9; Applied Biosystems). In real time PCR, different thermal profiles may be established for the assays.
  • DNA could be extracted before analysis, e.g. with a QIAamp Blood Kit (Qiagen). After, determining the size distribution, it is assessed whether the size distribution shows an increased non-apoptosis peak relative to a control, which would be is indicative of the disease.
  • QIAamp Blood Kit Qiagen
  • the plasma DNA size distribution shows one peak representing apoptotic cells (Diehl et al, 2005; Lo et al, 2010).
  • the peak is therefore referred to as apoptosis peak. It is characterized by DNA fragments of about 166 bp.
  • the non-apoptosis peak is a further (or second) peak in the size distribution which usually represents larger DNA fragments than the apoptosis peak.
  • the second peak is referred to as the "non- apoptosis peak” (which means that it is not the apoptosis peak) or "viability peak” (as it does not predominately represent apoptotic cells).
  • the peak is to be regarded as increased, if its size is increased.
  • An increase may be an increased area underneath the part of the graph which is considered to constitute the peak, i.e. the area integral of the peak.
  • the maximum of the peak i.e. the height of the peak, may be increased. It is evident that the increase in comparison to the control should be significant.
  • the person skilled in the art knows statistical procedures to assess whether two values or areas are significantly different from each other such as Student's t-test or chi-square tests. Based on this, a threshold may be determined by the skilled person, which will evidently depend from the method used. If a value (e.g. peak area or maximum) is at or above that threshold, the value is to be regarded as increased relative to the control.
  • the increase is at least about 10 % relative to the control. In other embodiments, the increase is at least 20 %, 30 %, 40 %, 50 % or 100 %, especially 150 %, 200 %, 250 %, or 300 %.
  • the control may be a sample from a healthy subject or determined at a group of healthy subjects. Alternatively, it may be a known and/or pre-determined reference value or area or size distribution. Furthermore, the skilled person knows how to select a suitable control.
  • an increased non-apoptosis peak is indicative of the disease. Accordingly, it can be assumed that the subject (or in case of a pregnant women the fetus) whose plasma DNA is analyzed is diseased, if the peak is increased. Further, the DNA comprised under (represented by) the non-apoptosis peak provides information useful in characterizing the disease.
  • the disease may particularly be a disease involving genomic aberrations, particularly chromosome aberrations (e.g. cancer or fetal aneuploidy).
  • a genomic abnormality or aberration is a change in the genome of a subject, i.e. the genome of one or more cells or all cells of the subject.
  • Down Syndrome also known as Trisomy 21 (an individual with Down Syndrome has three copies of chromosome 21 or a critical portion thereof, rather than two).
  • Turner Syndrome is an example of a monosomy where the individual is born with only one X-chromosome, also referred to as an X0 genotype.
  • Structural abnormalities are those abnormalities in which the chromosome's structure is altered. This can take several forms including deletions, duplications, translocations, inversions, rings and isochromosomes. In deletions, a portion of the affected chromosome is missing or deleted.
  • Known disorders in humans include Wolf-Hirschhorn syndrome, which is caused by partial deletion of the short arm of chromosome 4; and Jacobsen syndrome, also called the terminal 1 lq deletion disorder.
  • duplications a portion of the chromosome is duplicated, resulting in extra genetic material.
  • Known human disorders include Charcot-Marie-Tooth disease type 1A which may be caused by duplication of the gene encoding peripheral myelin protein 22 (PMP22) on chromosome 17.
  • PMP22 peripheral myelin protein 22
  • translocations a portion of one chromosome is transferred to another chromosome. There are two main types of translocations. In a reciprocal translocation, segments from two different chromosomes have been exchanged.
  • the genomic abnormality or aberration may be a mutation which is a change in the genomic sequence (here the DNA sequence). Mutations are caused by radiation, viruses, transposons and mutagenic chemicals, as well as errors that occur during meiosis or DNA replication. They can also be induced by the organism itself, by cellular processes such as hypermutation.
  • the sequence of a gene can be altered in a number of ways. Gene mutations have varying effects on health depending on where they occur and whether they alter the function of essential proteins.
  • Mutations in the structure of genes can be classified as small-scale mutations, such as point mutations, missense mutations, nonsense mutations, insertions, deletions and large-scale mutations in chromosomal structure, including amplifications (or gene duplications), deletions of large chromosomal regions, chromosomal translocations, interstitial deletions, chromosomal inversions, or loss of heterozygosity.
  • Changes in DNA caused by mutation can cause errors in protein sequence, creating partially or completely non- functional proteins. To function correctly, each cell depends on thousands of proteins to function in the right places at the right times. When a mutation alters a protein that plays a critical role in the body, a medical condition can result.
  • the size distribution of the plasma-DNA is detected as being biphasic, wherein the first and second peaks are the apoptosis peak and the non-apoptosis peak, respectively.
  • apoptosis peak in the size distribution of the plasma-DNA.
  • a peak means that there is a significant accumulation of DNA in a particular size range as compared to the surrounding part(s).
  • the apoptosis peak is typically in the size range of from 80 bp to 240 bp with a maximum around 166 bp. Also typically the peak is bell-shaped. It reflects the apoptotic cells (see above).
  • non- apoptosis a second peak, referred to as the “non- apoptosis” or “viability peak”. This peak typically immediately follows the apoptosis peak and has an asymmetric shape.
  • the non-apoptosis peak may be characterized by further details, particularly
  • DNA in the size of above 200 bp, particularly in the range of from 200 bp to about 1000 bp, more particularly of from 240 bp to 600 bp, especially from 250 bp to 400 bp.
  • the apoptosis peak may be characterized by further details, particularly
  • DNA - representing DNA in the range of from 50 bp to 250 bp, more particularly from 80 to 240 bp.
  • an increased non-apoptosis peak is indicative of a disease.
  • the non-apoptosis peak is understood as being increased
  • the non-apoptosis peak contains at least 5 % of the total plasma-DNA, particularly at least 10 %, more particularly at least 20 %; and /or
  • the ratio of the maximal height of the non-apoptosis peak to the maximal height of the apoptosis peak is at least 20 %, preferably at least 30 %, more preferable at least 33 %.
  • An increased non-apoptosis peak may also be detected based on its effect within the size distribution of one sample. As detailed above, the non-apoptosis peak is absent in the sample of a healthy subject. The presence of the peak requires that a particular fraction of the total DNA represented by the peak. Accordingly, the peak is increased if it contains at least 5 % of the total plasma-DNA, particularly at least 10 %, more particularly ate least 20 %.
  • the increased peak may also be detected based in its relation to the apoptosis peak.
  • the non-apoptosis peak is increased, if the ratio of the maximal height of the non-apoptosis peak to the maximal height of the apoptosis peak is at least 20 %, preferably at least 30 %, more preferable at least 33 %.
  • the method of the present invention may comprise a further diagnostic step, particularly if an increased non-apoptosis peak in the size distribution relative to a control is detected.
  • the present invention may provide a two-step approach, wherein first a biphasic size distribution of the plasma DNA is established and second a further diagnostic process carried out. Particular reference is made to the flow sheet presented in Figure 4.
  • the first step allows an efficient and low-cost pre-selection of individuals which are subsequently subjected to more specific testing.
  • detection of circulating tumor cells (CTCs) and optionally subsequent correlation of the biphasic size distribution with the presence of CTCs is envisioned.
  • CTCs circulating tumor cells
  • the method of the present invention provides the means to re-constract the tumor genome using the DNA of the non-apoptotic peak, particularly in the case of a cancer patient.
  • the concept aims at detecting in this DNA fraction genetic aberrations indicative of disease status and allowing to make therapeutic decisions.
  • DNA of the non-apoptotic peak can be used in array-based analysis.
  • genome-wide copy number changes can be established. Accordingly, the method allows to establish genome-wide copy number changes in the genome of the patient to be diagnosed, particularly the genome of a cancer patient or the genome of the fetus in a pregnant female, from plasma DNA; and/or to provide information about genomic aberrations such as copy numbers of the genome from the plasma DNA.
  • the determining of the size distribution may be done by any suitable means (see also above).
  • Micro fluidics-based electrophoresis has been shown as particular suitable.
  • electrophoresis which relies on inducing detectable differences in migration behavior between charged species under the influence of an applied electric field, has proven to be a highly versatile analytical technique owing to a favorable combination of characteristics including relatively simple hardware design and compatibility with a wide range of analytes including biological macro molecules (e.g., DNA, proteins). Therefore, it is a suitable tool for determining the size of DNA.
  • electrophoresis technology has been miniaturized into microfluidic formats with the aim of producing portable low-cost versions of conventional benchtop-scale instrumentation.
  • Microfluidic device can usually be identified by the fact that it has one or more channels with at least one dimension less than 1 mm.
  • Common fluids used in microfluidic devices include whole blood samples, bacterial cell suspensions, protein or antibody solutions and various buffers. Because the volume of fluids within these channels is very small, usually several nanoliters, the amount of reagents and analytes used is quite small.
  • An exemplary suitable device is the Agilent 2100 Bioanalyzer which allows for automatically sizing and quantitating DNA samples (Agilent Technologies, Inc., Santa Clara, CA, USA).
  • the method further comprises determining the total plasma-DNA level, wherein an increased total plasma-DNA level is also indicative of the disease.
  • a control e.g. a healthy subject
  • DNA of apoptotic cells is released into the blood (Leon at al, 1977, Stroun et al, 1989, Diehl et al, 2005). This has also been confirmed in the Examples (see e.g. Figure 1). Accordingly, the determination of total plasma-DNA may also be part of the method of the present invention. Total plasma-DNA may be determined as known in the art.
  • total plasma-DNA may be determined as the area integral of the size distribution, i.e. the area underneath the complete graph. It is evident that the increase in comparison to the control should be significant. The person skilled in the art knows statistical procedures to assess whether two values or areas are significantly different from each other such as Student's t-test or chi-square tests. It is evident for the skilled person that any background signal has to be subtracted when analyzing the data.
  • the increase is at least about 10 %. In other embodiments, the increase is at least 20 %, 30 %, 40 %, 50 % or 100 %, especially 150 % or 200 %.
  • the method of the present invention may comprise further steps.
  • the method may further comprise amplifying the plasma-DNA and analyzing the amplified DNA, particularly by comparative genomic hybridization (CGH), especially by microarray-based CGH.
  • CGH comparative genomic hybridization
  • CGH is a molecular-cytogenetic method for the analysis of copy number changes (gains/losses) in the DNA content of a given subject's DNA.
  • CGH can detect unbalanced chromosomal changes.
  • DNA from a subject and from a control (or reference) are each labeled with different tags for later analysis by fluorescence.
  • unlabeled human cot-1 DNA placental DNA that is enriched for repetitive DNA sequences such as the Alu and Kpn family
  • the mix is hybridized to normal metaphase chromosomes or, for microarray- or matrix-CGH, to an array or matrix containing hundreds or thousands of defined DNA probes.
  • Chromosomal CGH is capable of detecting loss, gain and amplification of the copy number at the levels of chromosomes.
  • plasma DNA is amplified before CGH according to method known by the skilled person. Exemplary methods for amplification and CGH are also detailed in the Examples.
  • a highly preferred protocol is detailed in the following: The laboratory part comprises a clearly defined protocol, specifying how to prepare plasma DNA and the parameters that may be used to diagnose a disease, necessary method steps and further suitable analyses.
  • Preparation from plasma DNA from a blood sample This step may be carried as follows: 80-90% of the plasma DNA of a healthy human is derived from about hematopoietic cells and 10-20% is derived from non- hematopoietic cells. As the total amount of plasma DNA is usually increased in tumor patients and pregnant females, the ratios may vary as the additional DNA results from tumor or fetal cells. It is evident for the skilled person that during the preparation of DNA, enrichment of hematopoietic cells should be avoided. This may be achieved by using specific tubes for collecting blood and preparation techniques which may include the addition of formaldehyde in order to stabilize the cell membranes of blood cells in order to reduce lysis. Evidently, it is also advantageous that plasma rather than serum is used.
  • the first region reflects enrichment at about 166 bp, which is referred to as the "apoptosis peak". If the plasma-DNA is, for example, assessed by a bioanalyzer, a size distribution of DNA may be shown on the X axis in the unit base pairs.
  • the apoptosis peak is usually obtained as a bell-shaped curve typically for a Gaussian distribution, wherein the maximum is usually at about 166 base pairs (which can vary by several base pairs).
  • a nucleosome is a complex of DNA and histones.
  • a nucleosome core consists of two exemplars of each histone H2a, H2b, H3 and H4 which is surrounded by 1.65 coils of 147 bp DNA.
  • the area between two linkers has a length of 50-60 base pairs in humans. According to established theories, the DNA of tumor cells or fetal cells is predominantly derived from apoptotic cells.
  • DNAse enzyme cleaves genomic DNA at internucleosomal regions resulting in fragments of 147 to 200 bp with a peak usually at about 166 base pairs.
  • the assessment of the regions allows for a first conclusion.
  • the second area consists of DNA fragments having more than 300 base pairs.
  • the size distribution of these areas does usually not show a Gaussian distribution curve, but may be asymmetric, usually with a steep increase to a maximum at about 308 base pairs, followed by a slow decrease at larger DNA fragments which might be until 10,000 bp in size.
  • the DNA fragments having a length of about 300 base pairs is composed of DNA of two subsequent nucleosomes. Larger fragments reflect the corresponding longer DNA molecules, such as several subsequent nucleosomes.
  • the second peak reflects therefore the extent to which the DNA was digested by DNAse and, therefore, whether or not the DNA is derived from apoptotic cells only, or a combination of apoptotic cells and viable cells. Therefore, the peak is referred to as "viability peak” or "non-apoptotic peak”.
  • the heights of the viability peaks have always been lower than those of the apoptosis peaks and the ratio of the prevailing heights was often in the range of about 6: 1 (apoptosis peak: non-apoptosis peak); however, this ratio is not fixed and may vary.
  • the viability peak correlates also with the occurrence of circulating tumor cells (CTCs).
  • apoptosis and viability peaks it is decided upon the further process, e.g., whether copy numbers of tumor cells are reflected in plasma DNA in an appropriate manner and whether plasma DNA should be further analyzed, whether sequencing of plasma DNA should be contemplated and whether CTCs are probably to be found, etc. Accordingly, further tests may be added in order to increase the informative value of the results of the size distribution. Threshold values may be determined for apoptosis and viability peaks which are to be reached in order to provoke further procedure steps. 5. Thereafter, a DNA library is prepared (e.g., by adding linkers to the ends of the DNA fragments), thereafter the fragments are amplified by PCR. A commercial kit such as WGA2-kit (Sigma- Aldrich Chemie GmbH, Kunststoff, Germany) may be used.
  • the size distribution of the amplification product may be again established and depending on the size of the fragments of plasma DNA a further amplification may be carried out.
  • the amplification product may be analyzed by different methods such as CGH (comparative genomic hybridization), particularly array CGH.
  • CGH comparative genomic hybridization
  • array CGH complementary genomic hybridization
  • the amplification product and the reference DNA, each labeled with different fluorochromes, are immobilized on an array by hybridization to immobilize sequences, whose physical address in the genome is known. Accordingly, a high resolution analysis of the copy number of the plasma DNA may be reached (similar techniques are detailed in Fiegler et al., 2007; Geigl et al, 2009).
  • the algorithm for array CGH as detailed at Geigl et al, 2009 has been completely revised and new properties have been added.
  • the algorithm identifies and corrects automatically common artefacts which may occur due to strong fragmentation of the plasma DNA.
  • the calculation to identify over- and underrepresented regions was completely revised.
  • a specific calculation procedure in order to establish copy numbers of chromosomes 13, 18, and 21 was added.
  • the prepared plasma DNA may be sequenced by next-generation sequencing or individual genes may be analyzed by Sanger sequencing or other procedures such as the SNaPshot assay (Dias- Santagata et al, 2010).
  • plasma DNA fragments might be further analyzed by deep sequencing in order to detect MRDs.
  • the method of the present invention is intended for the diagnosis of a disease.
  • the method is particularly suitable for a disease associated with increased apoptosis, particularly wherein the disease is cancer or fetal aneuploidy.
  • Apoptosis is the process of programmed cell death (PCD) that may occur in multicellular organisms. Biochemical events lead to characteristic cell changes (morphology) and death. These changes include blebbing, cell shrinkage, nuclear fragmentation, chromatin condensation, and chromosomal DNA fragmentation. Altered apoptosis can result in a number of cancers, autoimmune diseases, inflammatory diseases, and viral infections. Loss of control of cell death can lead to neurodegenerative diseases, hematologic diseases, and tissue damage.
  • the disease to be diagnosed is cancer or fetal aneuploidy.
  • Cancer is a term for a large group of different diseases. Cancers create malignant tumors, cells that divide and grow uncontrollably and invade nearby parts of the body. The cancer may also spread to more distant parts of the body through the lymphatic system or bloodstream. Not all tumors are cancerous. Benign tumors do not grow uncontrollably, do not invade neighbouring tissues, and do not spread through the body. Cancer is fundamentally a disease of failure of regulation of tissue growth. In order for a normal cell to transform into a cancer cell, the genes which regulate cell growth and differentiation must be altered. In general, the method of the present invention is applicable to any cancer associated with increased apoptosis and genetic aberration. In particular, inventors were able to detect colorectal cancer, breast cancer, prostate cancer, and lung cancer with the method of the present invention.
  • the method of the present invention further comprises the step of detecting the presence of CTCs, particularly if the non- apoptosis peak is increased. It could be shown that an increased non-apoptosis peak may be associated with an increased number of CTCs and that the size of the non-apoptosis peak may be correlated with the number of CTCs.
  • CTCs are cells that have detached from a primary tumor and circulate in the bloodstream are therefore an indicator for cancer. Cancer research has demonstrated the critical role circulating tumor cells play in the metastatic spread of carcinomas and CTCs may constitute seeds for subsequent growth of additional tumors (metastasis) in different tissues. Methods for detecting CTCs with the requisite sensitivity and reproducibility in patients with metastatic disease are known in the art.
  • Fetal aneuploidy is an aneuploidy (see above) in an unborn child. Fetal aneuploidy may be detected by analyzing the mother's plasma DNA which also comprises fetal DNA (see below).
  • the methods of the present invention may be used in the diagnosis of a disease, wherein the diagnosis may be a predictive or prognostic diagnosis or therapy monitoring, particularly of cancer.
  • Prognostic bio markers can be separated in two groups: bio markers that give information on recurrence in patients who receive curative treatment and biomarkers that correlate with the duration of (progression free) survival in patients with metastatic disease. According to a NIH Consensus Conference, a clinical useful prognostic marker must be a proven independent, significant factor that is easy to determine and interpret and has therapeutic consequences.
  • a biomarker with predictive value gives information on the effect of a therapeutic intervention in a patient.
  • a predictive biomarker can also be a target for therapy. One can distinguish upfront and early predictive markers. The first can be used for patient selection and the second provides information early during therapy (see Oldenhuis, 2008).
  • the plasma may be obtained from any suitable subject, as also detailed above.
  • the subject is a mammal.
  • subjects of particular interest include domestic animals, pets, and animals of commercial value (e.g. domestic animals such as horses) or personal value (e.g. pets such as dogs, cats).
  • the method is especially preferred with human subjects, for which diagnostic methods including pre-natal diagnostic methods are commonly employed.
  • the method of the present invention may also be used in the detection of a fetal disease, wherein the plasma sample is from the pregnant mother.
  • the plasma sample is from the pregnant mother.
  • the plasma sample is from the pregnant mother's blood if the disease is aneuploidy of the respective fetus.
  • trisomies 13, 18 and 21 are of clinical relevance. Of these, trisomy 21 and trisomy 18 are the most common.
  • a fetus with trisomy 13 can survive. Accordingly, if an increased non-apoptosis peak is identified in the plasma distribution, preferably the fetus or fetal DNA is further tested for trisomy 13, 18 and/or 21.
  • Quantification of fetal DNA is typically based on Y chromosome specific sequences. In this case, analysis is limited to pregnancies carrying a male fetus.
  • qRT-PCR of the y-specific SRY gene and a housekeeping gene, e.g. GAPDH or ⁇ -globin can be used.
  • GAPDH or ⁇ -globin a housekeeping gene, e.g. GAPDH or ⁇ -globin
  • Genomic male DNA was diluted to concentrations of 66 ng, 6 ng, 660 pg, 66 pg, 6.6 pg and run in parallel and in duplicates with each analysis.
  • the amount of 6.6 pg of DNA corresponds to one genome equivalent (GE) representing the DNA content of one single cell and was used for calculation for copy numbers of the specific target gene.
  • GE genome equivalent
  • the concentration, expressed in copies per milliliter, was calculated using the following equation: c Q x (VDNA/VPCR) X ( 1/V ext) [c, concentration in copies/ml; Q, target quantity (GE); V DNA , total volume of DNA obtained after extraction, V PCR , volume of input DNA for PCR, V ex t volume of plasma extracted] Because the SRY gene is found in all nucleated cells of males only, whereas the ⁇ -globin gene is present in the male fetus and the pregnant female, we calculated the percentage of male DNA in a particular plasma sample, denoted as Y%, using the following equation (Lui et al, 2002):
  • DNA-Methylation relates to the presence of methyl groups at the 5' carbon atom of a cytosin following a guanosin (a so- called CpG dinucleotide). CpG-Methylation in the promoter regions of genes is part of the regulatory system of gene expression and is tissue-specific.
  • genes may be selected for analysis of fetal cells in maternal blood, whose methylation differs between fetal cells (usually placenta cells as the majority of fetal cells in maternal blood in derived from the placenta) and maternal blood cells (Chiu RW, Lo YM (2011)).
  • the methylation profile of the SERPINB5 (Serpin peptidase inhibitor, clade B (ovalbumin), member 5) promoter may be used, which is hypomethylated and hypermethylated in fetal and maternal cells, respectively (Chim SS, et al (2005)).
  • a 5- methylcytidine-specific antibody may be used to detect methylated sequences and to enrich for fetal methylated DNA (see Papageorgiou EA, et al (2011)).
  • the present invention relates to the use of the size distribution of plasma DNA in the diagnosis of a disease, wherein an increased non-apoptosis peak in the size distribution relative to a control is indicative of the disease.
  • the present invention relates to the use of size distribution of plasma DNA in the diagnosis of a disease associated with increased apoptosis, wherein the size distribution is biphasic having a apoptosis peak and a non-apoptosis peak, wherein the apoptosis peak is characterized by a maximum in the range of from 150 bp to 180 bp and wherein the non-apoptosis peak is characterized by a maximum at in the range of from 300 bp to 350 bp, and wherein an increased non-apoptosis peak in the size distribution relative to a control is indicative of the disease.
  • Plasma-DNA analysis from patient 22 (l .Peak: 81-241 bp and 67 %; 2. Peak: 249-400 bp and 25 %).
  • Patients with the second, non-apoptotic peak (2) have higher plasma-DNA concentrations (mean: 604 ng/ml; median: 562 ng/ml; range: 260-1037 ng/ml) as compared to the patients without this second peak (1) (mean: 103 ng/ml; median: 89 ng/ml; range: 22-201 ng/ml) ( O.0001).
  • Figure 2 shows a combined copy number profile representing an average profile from all individual CTCs.
  • Figure 3 shows exemplary plasma-DNA size distributions, (a) Smear analysis with the 2100 Expert software of the plasma-DNA of healthy control M3. Sliders were placed at the beginning and end of the peak to determine the % of total, (b) Smear analysis of patient #18 (Mini Kit, 800 pg) with sliders positioned at the first (101 bp - 247 bp) and second (254 bp - 590 bp) peak.
  • Figure 4 shows the workflow of a preferred method of the present invention.
  • CTCs plasma-DNA and circulating tumor cells
  • Group a) contains one patient (i.e. #38), who was initially treated with surgery alone; however, progression was noted 44 months later. Therefore, this patient is not "newly diagnosed", but he did not receive any chemotherapy as patients in groups b) and c) prior to our blood collection and therefore he was included in group a).
  • the interval between the last chemotherapy and blood collection depended on the above definition of the three different groups with progressive disease and did accordingly not exist for group a) (the mean interval between the diagnosis of the primary and the blood collection was for this group: 8.5 months; median: 1 months; range: 1-45 months; this extensive range was caused by patient #38, as mentioned above).
  • the mean interval between last chemotherapy and our blood collection was 198 days (median: 121 days; range: 67-688 days) for group b) (Interval between diagnosis primary and blood collection: mean: 49 months; median: 41 months; range: 8-151 months); and 25 days (median: 21 days; range: 12-48 days) for group c) (Interval between diagnosis primary and blood collection: mean: 20 months; median: 16 months; range: 2-47 months).
  • DNA was isolated from plasma samples using the QIAamp DNA Blood Mini Kit (#51306, Qiagen, Hilden, Germany) or the Qiagen circulating nucleic acids Kit (Qiagen, Hilden, Germany) according to the manufacturer's instructions. DNA was eluted in 30 of distilled water. Quantification and quality of the extracted DNA were determined using the Nano-Drop Spectrometer ND- 1000 (Peqlab Biotechnologie, Er Weg, Germany).
  • Quant-iTTM PicoGreen ® dsDNA Kit (Invitrogen, Carlsbad, CA, USA) according to the manufacturer ' s instructions. Briefly, for a standard curve (ranging from 500pg ⁇ l to 16pg ⁇ l) we used bacteriophage lambda DNA. 50 ⁇ 1 of the standard dilutions were transferred into disposable cuvettes and mixed with 50 ⁇ 1 of Quant-iTTM PicoGreen® reagent working solution. Experimental DNA samples were diluted in lxTE (1 :25) to a final volume of 50 ⁇ 1 and also mixed with 50 ⁇ 1 of Quant-iTTM PicoGreen® reagent working solution in disposable cuvettes.
  • Fluorescence of the samples was then measured using a QuantiFluorTM Fluorometer (Promega, Madison, WI, USA). The fluorometer was calibrated using the blank and the highest concentration of the standard dilutions. DNA concentration of the samples was determined from the standard curve generated using a spreadsheet software such as Excel (Microsoft Corp.).
  • the minimal amount of plasma-DNA needed for the Bioanalyzer was defined by the amount of plasma-DNA from healthy donors needed to visualize the apoptosis peak. For each sample the same amount of DNA, i.e. 800 pg, was employed to reveal relative differences between the samples.
  • the length of the native plasma-DNA was determined by electrophoresis on an Agilent 2100 Bioanalyzer using the DNA series II 1000 kit / Agilent High Sensitivity DNA kit.
  • the Agilent 2100 Expert software (version B.02.07 or higher) offers a smear analysis with an integrator allowing size adjustments of the smear region.
  • the software automatically determines the average size (bp), size distribution in CV (%), concentration (pg/ ⁇ ), % of total, and molarity (pmol/L) for each defined smear region.
  • the Agilent 2100 Bioanalyzer allows smear analysis for DNA sizing and quantification. In each sample we focused on the range from 80 to 600 bp as we invariably observed merely a flat line without measurable fluorescence intensities below 80 bp or above 600 bp, indicating that no measurable DNA is present in this range.
  • the Agilent software determines the average size (bp), size distribution in CV (%) and % of total for each defined smear region.
  • DNA concentration was determined by a Nanodrop spectrophotometer. We used 50 ng of plasma-DNA as starting template for the whole genome amplification. After amplification the mean concentration of DNA was 136.013 ⁇ 4/ ⁇ 1 (min 57.32 ng/ ⁇ ; max 241.12 ng/ ⁇ ). DNA Isolation of tumor DNA from FFPE sections
  • FFPE Formalin- fixed paraffin embedded
  • the EpCAM-enriched cell fraction in a volume of 300 ⁇ was transferred from the CellSearch cartridge onto slides.
  • Nucleated CK-positive, CD45- negative CTCs were isolated with the help of a micromanipulation device comprising the microinjector CellTram vario and the micromanipulator Trans ferM an NK2 (Eppendorf AG Hamburg, Germany) connected to an Axiovert 200 inverted microscope (Carl Zeiss AG, Jena, Germany) Single CTCs were placed into a 2.5 ⁇ water drop in a 200 ⁇ PCR reaction tube and stored overnight at -20°C prior to whole genome amplification.
  • GenomePlex Single Cell Whole Genome Amplification Kit Sigma-Aldrich, St. Louis, MO, USA
  • GenomiPhi DNA amplification kit GE Healthcare, Chalfont St. Giles, UK
  • WGA products were purified using the GenElute PCR Clean-up Kit (Sigma-Aldrich, St. Louis, MO, USA) for DNA obtained with the GenomePlex Single Cell WGA Kit (Sigma- Aldrich) or NucleoSeq Columns (Macherey-Nagel, Diiren, Germany) for GenomiPhi amplified DNA. The final DNA concentration was determined by a NanoDrop ND-100 Spectrometer (Peqlab Biotechnologie, Er Weg, Germany).
  • Array-CGH was carried out using a genome-wide oligonucleotide microarray platform (Human genome CGH 60K microarray kit, Agilent Techologies, Santa Clara, CA, USA) following the instructions of the manufacturer (protocol version 6.0).
  • reference DNA we used commercially available male reference DNA (Promega, Madison, WI, USA), which was amplified for the hybridization with WGA2.
  • Samples were labeled with the Bioprime array-CGH genomic labeling system (Invitrogen, Carlsbad, CA, USA) according to the manufacturer's instructions.
  • 500 ng test DNA and reference DNA were differentially labeled with dCTP-Cy5 or dCTP-Cy3 (GE Healthcare, Milwaukee, WI, USA). Slides were scanned using a microarray scanner, and images were analyzed using Feature Extraction and DNA Workbench 5.0.14 (Agilent Technologies) with the statistical algorithm ADM-2; the sensitivity threshold was 6.0.
  • the algorithm calculates the mean ratio value for each window based on the respective ratio values. Assuming that a window's ratio values are distributed normally we estimate the standard deviation (SD) by considering the outmost value that is within ⁇ 34.1% of the mean. Thresholds were defined as ⁇ 1.25 times the SD in single cell experiments and due to the higher noise with plasma-DNA thresholds had to be defined more stringently as ⁇ 1.5 times the SD. The obtained values were assigned to all oligos of the respective window. If a window shows a significantly increased or decreased mean ratio value the mean position of that window will be displayed above or below the respective region of the ratio profile. We used 7 different window sizes consisting of 10, 25, 50, 100, 250, 500 and 750 adjacent oligos.
  • the assessment of the copy number status of each oligo is based on 44 different calculations. The final assessment is indicated in a single green or red bar for gained or lost, respectively, regions, which is generated if at least 39 (90%) of the 44 repetitive calculations consistently result in the same copy number change. Furthermore, the algorithm generates a table with all localizations of significant calls which allows detailed mapping of each CNV. All ratio profiles shown in the center of the images were calculated using a 500 oligonucleotide window.
  • Copy number estimations after WGA may have to deal with potential amplification artifacts, which may result in under- (e.g. allele drop out) or over-representations (e.g. preferential amplifications).
  • Such a systematic amplification bias was not observed with the single cell amplification products as reported before by us (Geigl et al. 2009; Geigl and Speicher 2009) and therefore we had not employed corrections for single cell analyses.
  • amplification biases and since a large part of this amplification bias was systematic (e.g. correlated with GC-content of the DNA) we could correct for it.
  • a first step we mapped the systematic amplification biases.
  • a second step we introduced corrections for these regions in our algorithm for plasma-DNA.
  • the regions found in the first step we increased the number of repetitive calculations, which had to consistently result in the same copy number change from 39 (90%) of the 44 calculations to 41 (93%) requesting that the copy number change is also indicated at least in the 500 or 750 window size calculation.
  • the ratio profile in the center of our array-CGH illustrations these regions were adjusted with a correction factor, which depended on the aforementioned calculations. The same corrections were applied in all plasma-DNA analyses of the tumor patients.
  • Single cells should have integer copy number states. We inferred them from the array- CGH ratio values as follows: A nullosomy is reflected by a log 2 (0) ratio value, which should in theory result in an infinite small ratio value, which is in practice usually indicated by a very small ( ⁇ -2) log 2 ratio value. A heterozygous deletion in a diploid cell results in a ratio of 1 :2, which translates to a log 2 (0.5) ratio value of -1. With the exception of nullosomies there are in a triploid cell two (i.e. 2:3; log 2 ratio: -0.585 and 1 :3; log 2 ratio: - 1.585) and in a tetraploid cell three (i.e.
  • array-CGH offers the option to compare the respective array-CGH ratio profile with the integer copy number profile.
  • a standard shotgun library was made from genomic (tumor and metastasis of patients 6 and 26) or amplified (CTCs from patients 6 and 26) DNA.
  • Four libraries were pooled equimolarily (250 ng each) (FFPE pool: tumor and metastasis of patient 6 and 26, CTC pool 1 : CTC 7, 13, 14 from patient 6, CTC 5 from patient 26; CTC pool 2: CTC 21 , 22, 24, 28 from patient 26) and hybridized to the SeqCap EZ Oligo pool for 72h. Streptavidin beads were used to pull down the complex of captured oligos and genomic DNA fragments, whereas unbound fragments were removed by washing. Finally, the enriched fragment pools were amplified by PCR. For quality control of enrichment qPCR at control loci included in the Choice Library and for one target gene (TP53) was performed. Average fold-enrichment for control loci enriched was 4321 -fold and for the selected target gene TP53 9210-fold.
  • enriched library pools were ready for high throughput (next generation) sequencing.
  • KRAS KRAS specific length 119bp, 168bp, and 323bp
  • Primers were designed resulting in 3 different amplicon lengths (KRAS specific length 119bp, 168bp, and 323bp), including the Roche-compatible adaptors A and B with a length of 21bp, plus 4bp TCAG key sequence (each read has to start with key sequence) and lObp MID (multiple identifier; barcode).
  • Amplicons from patient and control samples were purified, quantified with PicoGreen, and pooled equimolarily (6-8 samples per pool). Amplicon pools were quality checked on an Agilent Bioanalyzer using Agilent High Sensitivity DNA kit.
  • Emulsion PCR was performed according to emPCR Method Manual - Lib-A MV. In doing so, two emulsion PCRs were performed for forward and reverse sequencing. After clonal amplification microbeads were collected and DNA-carrying beads were enriched and deposited onto PicoTiterPlates provided for the 454-FLX instrument (Roche Diagnostics).
  • Massively parallel pyrosequencing was performed according to the manufacturer's protocol, base calls and quality scores were generated using the GS Run Processor software on a HPC-cluster, and variants were extracted using the GS Amplicon Variant Analysis 2.6 software provided with the platform. Read lengths strongly corresponded to amplicon lengths. The average coverage was 57045x for the 119bp fragment, 12201x for the 168bp fragment, and 13540x for the 323bp fragment, respectively.
  • Table 1 Ultra-deep pyrosequencing with sequencing reaction sizes of 119 bp, 168 bp, and 323 bp, in patients without non-apoptosis peak (7, 11, 15, and 16) and with non-apoptosis peak (6, 10, 25, 38).
  • Deep sequencing was performed in four patients without (i.e. #7, #11 , #15, and #16) and in four patients with the non-apoptosis peak (i.e. #6, #10, #25, and #38).
  • patients without non-apoptosis peak deep sequencing identified mutated KRAS fragments at low levels in two patients (#11, #15), which was within the range of false-positives, and in the other two patients (#7, #16) even not at all (Table 1).
  • Table 1 shows that these patients may have longer DNA fragments in their plasma than healthy controls.
  • non-apoptotic cell plasma-DNA peak correlates with higher plasma- DNA concentrations and genomic plasma-DNA imbalances
  • patients with the second, non-apoptotic peak had higher plasma-DNA concentrations (mean: 604 ng/ml; median: 562 ng/ml; range: 260-1037 ng/ml) as compared to the patients without this second peak (mean: 103 ng/ml; median: 89 ng/ml; range: 22-201 ng/ml) ( O.0001) (Fig. 1 e)).
  • the plasma-DNA in 10 of the 11 patients i.e. 6, 9, 10, 18, 20, 22, 25, 26, 27, 33; exception: 38
  • the additional non-apoptosis peak had a mean number of oligonucleotides with copy number changes of 12.476 (22.3%; range: 9.117-14.915; 16.3%-26.7%) which was highly significant compared to both the aforementioned CRC cases and the healthy controls ( ⁇ 0.0001 each).
  • the chromosomal imbalances in the plasma-DNA are tumor specific
  • the primary tumor and its brain metastasis showed marked copy number differences, however, in the plasma-DNA we could attribute many chromosomal regions to the primary tumor and/or metastasis by shared copy number alterations.
  • the copy number status of 46.3% of the oligonucleotides on our array platform was omnipresent across all three lesions; 18.1% was partially shared by the primary tumor and 18.0% by the metastasis and 17.6% unique to the plasma-DNA.
  • CTC 4 had very similar copy number changes as to those in both primary and metastasis; however, in these CTCs the overrepresentation of chromosome 3, which was present in both the primary and metastasis was lost.
  • CTC2 had a private pattern of aberrations.
  • Veridex system identified 6 cells. However, none of them had the G12D KRAS mutation, which had been identified in the primary tumor and which we had used for deep sequencing. Moreover, all of them had a balanced profile suggesting that these cells were likely not tumor cells but epithelial stromal cells.
  • APC Adenomatous polyposis coli NAV3 Neuron navigator 3
  • Olfactory receptor family 51
  • CDKN2A inhibitor 2A (melanoma, pl6, OR51E1
  • subfamily E member 1 inhibits CDK
  • inhibitor 2A catalytic, alpha polypeptide
  • beta 1 increased 2 (S. cerevisiae)
  • viral oncogene homolog beta receptor II 70/80kDa
  • LAMA1 Laminin, alpha 1 TGM3 polypeptide, protein-glutamine- gamma-glutamyltransferase
  • nonpolyposis type 2 (E. coli)
  • lung metastasis may have a filtering capacity reducing both the number of mutant DNA fragments and CTCs is at present very speculative and warrants further investigations.
  • Table 5 Sites of metastases and their association with the non-apoptosis peak and number ofCTCs.
  • a major goal of cancer medicine is to progress from fixed treatment regimens to bespoke therapy tailored to a patient's tumor. Efficient monitoring response to anti-cancer therapy is a prerequisite for individualizing treatment choices.
  • Efficient monitoring response to anti-cancer therapy is a prerequisite for individualizing treatment choices.
  • the first group has elevated plasma-DNA levels compared to healthy controls, but as confirmed by deep sequencing and array-CGH a very low percentage of mutated DNA fragments. This is consistent with necrotic neoplastic cells being engulfed by macrophages, which involves the killing of neoplastic cells and surrounding stromal and inflammatory cells (Diehl et al. 2005).
  • the released DNA will contain multiple wild type DNA sequences and may thus explain the increase in total, non-mutant circulating DNA.
  • the second group is characterized by the non-apoptosis peak, which indicates a distinct biological process because its occurrence was associated with very high plasma-DNA levels, elevated percentages of mutated DNA fragments in the circulation and an increased number of CTCs.
  • the non-apoptosis peak likely reflects massive cell destruction with direct shedding of DNA from tumor cells and cellular fragments into the bloodstream. Schwarzenbach et al. (2009) suggested a link between the presence of CTCs and allelic imbalances of 3 microsatellite markers in patients with prostate cancer.
  • CTCs reflect a significant heterogeneity of the tumor fulfilling all criteria for chromosomal instability (Geigl et al. 2008) and thus provide unique insights into the tumor cell population substructure.
  • results from the plasma-DNA may reflect more the most predominant changes of the tumor genome at the time of blood collection. The ease with which plasma-DNA can be analyzed may make it to an especially attractive tool for disease monitoring.
  • translocations monitored in hematologic diseases are well-established driver mutations whereas relapsing cells in solid tumors do not have to carry a driver rearrangement found in the primary tumor. Instead, it may be possible for a relapsing clone to have lost the rearrangement and still be malignant.
  • solid tumors will likely require a higher number of molecular targets for disease monitoring, which may make approaches providing data on the entire genome as presented here, especially attractive.
  • Plasma-DNA and CTCs may evolve to a routine laboratory test to detect the development of an aggressive tumor subclone.
  • Our analyses demonstrate that progression of chromosomal copy number changes and acquisition of novel driver mutations during tumor evolution can be established from both plasma-DNA and CTCs. This may pave the way for new options for disease monitoring and may represent another step in the progress to "personalized genomics".
  • Wood LD et al. (2007) The genomic landscapes of human breast and colorectal cancers. Science 318: 1108-13. Epub 2007 Oct 11.

Landscapes

  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Organic Chemistry (AREA)
  • Genetics & Genomics (AREA)
  • Analytical Chemistry (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • Engineering & Computer Science (AREA)
  • Pathology (AREA)
  • Immunology (AREA)
  • Microbiology (AREA)
  • Molecular Biology (AREA)
  • Biotechnology (AREA)
  • Biophysics (AREA)
  • Physics & Mathematics (AREA)
  • Biochemistry (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • General Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Hospice & Palliative Care (AREA)
  • Oncology (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

Abstract

La présente invention concerne une méthode pour diagnostiquer une maladie, particulièrement une maladie associée à un renforcement de l'apoptose, et l'utilisation de la distribution de la taille de l'ADN plasmatique dans le diagnostic de la maladie.
PCT/EP2012/071117 2011-10-25 2012-10-25 Méthode pour diagnostiquer une maladie basée sur la distribution de l'adn plasmatique WO2013060762A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
EP12780167.8A EP2771483A1 (fr) 2011-10-25 2012-10-25 Méthode pour diagnostiquer une maladie basée sur la distribution de l'adn plasmatique

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
EP11008550 2011-10-25
EP11008550.3 2011-10-25

Publications (1)

Publication Number Publication Date
WO2013060762A1 true WO2013060762A1 (fr) 2013-05-02

Family

ID=47115898

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/EP2012/071117 WO2013060762A1 (fr) 2011-10-25 2012-10-25 Méthode pour diagnostiquer une maladie basée sur la distribution de l'adn plasmatique

Country Status (2)

Country Link
EP (1) EP2771483A1 (fr)
WO (1) WO2013060762A1 (fr)

Cited By (24)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2016008550A1 (fr) 2014-07-16 2016-01-21 Roche Diagnostics Gmbh Détection précoce non invasive de rejet de greffe d'organe solide par analyse quantitative d'amplicons du gène hla
EP3047038A1 (fr) * 2013-09-20 2016-07-27 The Chinese University Of Hong Kong Analyse par séquençage de l'adn circulant en vue de la détection et du suivi de maladies auto-immunes
CN107407626A (zh) * 2014-09-26 2017-11-28 加利福尼亚大学董事会 评估癌症的疾病状况的方法
US9850523B1 (en) 2016-09-30 2017-12-26 Guardant Health, Inc. Methods for multi-resolution analysis of cell-free nucleic acids
US10364467B2 (en) 2015-01-13 2019-07-30 The Chinese University Of Hong Kong Using size and number aberrations in plasma DNA for detecting cancer
US10453556B2 (en) 2015-07-23 2019-10-22 The Chinese University Of Hong Kong Analysis of fragmentation patterns of cell-free DNA
US10633713B2 (en) 2017-01-25 2020-04-28 The Chinese University Of Hong Kong Diagnostic applications using nucleic acid fragments
US10741270B2 (en) 2012-03-08 2020-08-11 The Chinese University Of Hong Kong Size-based analysis of cell-free tumor DNA for classifying level of cancer
WO2020190613A1 (fr) * 2019-03-15 2020-09-24 Stitch Bio, Llc Procédés de détection d'une maladie résiduelle minimale
WO2021023650A1 (fr) * 2019-08-02 2021-02-11 INSERM (Institut National de la Santé et de la Recherche Médicale) Procedes de depistage d'un cancer chez un sujet
JP2021509266A (ja) * 2017-12-19 2021-03-25 バイオロジカル ダイナミクス，インク． 無細胞ｄｎａ断片を検出および定量化するための方法とデバイス
US11062791B2 (en) 2016-09-30 2021-07-13 Guardant Health, Inc. Methods for multi-resolution analysis of cell-free nucleic acids
US11062789B2 (en) 2014-07-18 2021-07-13 The Chinese University Of Hong Kong Methylation pattern analysis of tissues in a DNA mixture
US11211144B2 (en) 2020-02-18 2021-12-28 Tempus Labs, Inc. Methods and systems for refining copy number variation in a liquid biopsy assay
US11211147B2 (en) 2020-02-18 2021-12-28 Tempus Labs, Inc. Estimation of circulating tumor fraction using off-target reads of targeted-panel sequencing
US11352670B2 (en) 2014-07-25 2022-06-07 University Of Washington Methods of determining tissues and/or cell types giving rise to cell-free DNA, and methods of identifying a disease or disorder using same
US11410750B2 (en) 2018-09-27 2022-08-09 Grail, Llc Methylation markers and targeted methylation probe panel
US11435339B2 (en) 2016-11-30 2022-09-06 The Chinese University Of Hong Kong Analysis of cell-free DNA in urine
US11459616B2 (en) 2016-10-24 2022-10-04 The Chinese University Of Hong Kong Methods and systems for tumor detection
US11475981B2 (en) 2020-02-18 2022-10-18 Tempus Labs, Inc. Methods and systems for dynamic variant thresholding in a liquid biopsy assay
US11534756B2 (en) 2016-03-24 2022-12-27 Biological Dynamics, Inc. Compact device for detection of nanoscale analytes
US11643693B2 (en) 2019-01-31 2023-05-09 Guardant Health, Inc. Compositions and methods for isolating cell-free DNA
US11731132B2 (en) 2017-12-19 2023-08-22 Biological Dynamics, Inc. Methods and devices for detection of multiple analytes from a biological sample
US11883833B2 (en) 2018-04-02 2024-01-30 Biological Dynamics, Inc. Dielectric materials

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2010033578A2 (fr) * 2008-09-20 2010-03-25 The Board Of Trustees Of The Leland Stanford Junior University Diagnostic non effractif d'aneuploïdie foetale par sequençage
WO2011054936A1 (fr) * 2009-11-06 2011-05-12 The Chinese University Of Hong Kong Analyse génomique basée sur la taille

Family Cites Families (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
NZ595993A (en) * 2009-03-31 2013-05-31 Oridis Biomarkers Gmbh Method for diagnosis of cancer and monitoring of cancer treatments
EP2426217A1 (fr) * 2010-09-03 2012-03-07 Centre National de la Recherche Scientifique (CNRS) Procédés analytiques pour acides nucléiques libres dans les cellules et applications

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2010033578A2 (fr) * 2008-09-20 2010-03-25 The Board Of Trustees Of The Leland Stanford Junior University Diagnostic non effractif d'aneuploïdie foetale par sequençage
WO2011054936A1 (fr) * 2009-11-06 2011-05-12 The Chinese University Of Hong Kong Analyse génomique basée sur la taille

Non-Patent Citations (56)

* Cited by examiner, † Cited by third party
Title
"R Development Core Team", R FOUNDATION FOR STATISTICAL COMPUTING, VIENNA, AUSTRIA, article "R: A language and environment for statistical computing"
ALEKSIC K ET AL.: "Evolution of genomic instability in diethylnitrosamine-induced hepatocarcinogenesis in mice", HEPATOLOGY, vol. 53, 2011, pages 895 - 904
BEGUS-NAHRMANN Y ET AL.: "p53 deletion impairs clearance of chromosomal-instable stem cells in aging telomere-dysfunctional mice", NAT GENET, vol. 41, 30 August 2009 (2009-08-30), pages 1138 - 43
BOYNTON K A ET AL: "DNA integrity as a potential marker for stool-based detection of colorectal cancer", CLINICAL CHEMISTRY, AMERICAN ASSOCIATION FOR CLINICAL CHEMISTRY, WASHINGTON, DC, vol. 49, no. 7, 1 July 2003 (2003-07-01), pages 1058 - 1065, XP002345190, ISSN: 0009-9147, DOI: 10.1373/49.7.1058 *
CHIM SS ET AL.: "Detection of the placental epigenetic signature of the maspin gene in maternal plasma", PROC NATL ACAD SCI USA, vol. 102, 3 October 2005 (2005-10-03), pages 14753 - 8, XP002355638, DOI: doi:10.1073/pnas.0503335102
CHIU RW ET AL.: "Non-invasive prenatal assessment of trisomy 21 by multiplexed maternal plasma DNA sequencing: large scale validity study", BMJ, vol. 342, 2001, pages C7401
CHIU RW; LO YM: "Non-invasive prenatal diagnosis by fetal nucleic acid analysis in maternal plasma: the coming of age", SEMIN FETAL NEONATAL MED, vol. 16, 12 November 2010 (2010-11-12), pages 88 - 93, XP028153879, DOI: doi:10.1016/j.siny.2010.10.003
CRISTOFANILLI M ET AL.: "Circulating tumor cells, disease progression, and survival in metastatic breast cancer", N ENGL J MED, vol. 351, 2004, pages 781 - 91, XP002458149, DOI: doi:10.1056/NEJMoa040766
DELIGEZER UGUR ET AL: "Size distribution of circulating cell-free DNA in sera of breast cancer patients in the course of adjuvant chemotherapy", CLINICAL CHEMISTRY AND LABORATORY MEDICINE, WALTER DE GRUYTER & CO, BERLIN, NEW YORK, vol. 46, no. 3, 1 January 2008 (2008-01-01), pages 311 - 317, XP009123312, ISSN: 1434-6621 *
DHALLAN R ET AL.: "A non-invasive test for prenatal diagnosis based on fetal DNA present in maternal blood: a preliminary study", LANCET, vol. 369, 2007, pages 474 - 81, XP005879775, DOI: doi:10.1016/S0140-6736(07)60115-9
DHALLAN R ET AL.: "Methods to increase the percentage of free fetal DNA recovered from the maternal circulation", JAMA, vol. 291, 2004, pages 1114 - 9, XP009076430, DOI: doi:10.1001/jama.291.9.1114
DIEHL F ET AL.: "Analysis of mutations in DNA isolated from plasma and stool of colorectal cancer patients", GASTROENTEROLOGY, vol. 135, 15 May 2008 (2008-05-15), pages 489 - 98
DIEHL F ET AL.: "Circulating mutant DNA to assess tumor dynamics", NAT MED, vol. 14, 31 July 2007 (2007-07-31), pages 985 - 90, XP002666722, DOI: doi:10.1038/NM.1789
DIEHL F ET AL.: "Detection and quantification of mutations in the plasma of patients with colorectal tumors", PROC NATL ACAD SCI USA, vol. 102, 28 October 2005 (2005-10-28), pages 16368 - 73, XP002518285, DOI: doi:10.1073/pnas.0507904102
DIEHL F ET AL: "Analysis of Mutations in DNA Isolated From Plasma and Stool of Colorectal Cancer Patients", GASTROENTEROLOGY, ELSEVIER, PHILADELPHIA, PA, vol. 135, no. 2, 1 August 2008 (2008-08-01), pages 489 - 498.E7, XP023901355, ISSN: 0016-5085, [retrieved on 20080801], DOI: 10.1053/J.GASTRO.2008.05.039 *
EISEN MB ET AL.: "Cluster analysis and display of genome-wide expression patterns", PROC NATL ACAD SCI USA, vol. 95, 1998, pages 14863 - 8, XP002939285, DOI: doi:10.1073/pnas.95.25.14863
FLOHR T: "Minimal residual disease-directed risk stratification using real-time quantitative PCR analysis of immunoglobulin and T-cell receptor gene rearrangements in the international multicenter trial AIEOP-BFM ALL 2000 for childhood acute lymphoblastic leukemia", LEUKEMIA, vol. 22, 31 January 2008 (2008-01-31), pages 771 - 82, XP055065436, DOI: doi:10.1038/leu.2008.5
GEIGL JB ET AL.: "Defining 'chromosomal instability", TRENDS GENET, vol. 24, 14 January 2008 (2008-01-14), pages 64 - 9, XP022441748, DOI: doi:10.1016/j.tig.2007.11.006
GEIGL JB ET AL.: "Identification of small gains and losses in single cells after whole genome amplification on tiling oligo arrays", NUCLEIC ACIDS RES, vol. 37, 18 June 2009 (2009-06-18), pages E105
GEIGL JB; SPEICHER MR: "Single-cell isolation from cell suspensions and whole genome amplification from single cells to provide templates for CGH analysis", NAT PROTOC, vol. 2, 2007, pages 3173 - 84, XP002667168, DOI: doi:10.1038/NPROT.2007.476
GEIGL JOCHEN B ET AL: "Single-cell isolation from cell suspensions and whole genome amplification from single cells to provide templates for CGH analysis", NATURE PROTOCOLS, NATURE PUBLISHING GROUP, GB, vol. 2, no. 12, 1 December 2007 (2007-12-01), pages 3173 - 3184, XP002667168, ISSN: 1750-2799, [retrieved on 20071206], DOI: 10.1038/NPROT.2007.476 *
GORMALLY E ET AL.: "Circulating free DNA in plasma or serum as biomarker of carcinogenesis: practical aspects and biological significance", MUTAT RES, vol. 635, 25 January 2007 (2007-01-25), pages 105 - 17
JABBOUR E ET AL.: "Molecular monitoring in chronic myeloid leukemia: response to tyrosine kinase inhibitors and prognostic implications", CANCER, vol. 112, 2008, pages 2112 - 8
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, AMERICAN ASSOCIATION FOR CANCER RESEARCH, US, vol. 61, no. 4, 15 February 2001 (2001-02-15), pages 1659 - 1665, XP002494286, ISSN: 0008-5472 *
KELVIN J LIU ET AL: "PCR-free, microfluidic single molecule analysis of circulating nucleic acids in lung cancer patient serum", ENGINEERING IN MEDICINE AND BIOLOGY SOCIETY,EMBC, 2011 ANNUAL INTERNATIONAL CONFERENCE OF THE IEEE, IEEE, 30 August 2011 (2011-08-30), pages 8392 - 8395, XP032027067, ISBN: 978-1-4244-4121-1, DOI: 10.1109/IEMBS.2011.6092070 *
LEARY RJ ET AL.: "Development of personalized tumor biomarkers using massively parallel sequencing", SCI TRANSL MED, vol. 2, 2010, pages 20RA14
LEON SA ET AL.: "Free DNA in the serum of cancer patients and the effect of therapy", CANCER RES, vol. 37, 1977, pages 646 - 50, XP001083834
LEWIN B: "Gene IX", 2008, pages: 757 - 795
LO Y M DENNIS ET AL: "Maternal plasma DNA sequencing reveals the genome-wide genetic and mutational profile of the fetus", SCIENCE / SCIENCE TRANSLATIONAL MEDICINE, WASHINGTON, DC : AAAS, US, vol. 2, no. 61, 8 December 2010 (2010-12-08), pages 61RA91 - 1, XP008132703, ISSN: 1946-6242, DOI: 10.1126/SCITRANSLMED.3001720 *
LO YM ET AL.: "Maternal plasma DNA sequencing reveals the genome-wide genetic and mutational profile of the fetus", SCI TRANSL MED, vol. 2, 2010, pages 61RA91
LO YM ET AL.: "Quantitative analysis of fetal DNA in maternal plasma and serum: implications for noninvasive prenatal diagnosis", AM J HUM GENET, vol. 62, 1998, pages 768 - 75, XP009002852, DOI: doi:10.1086/301800
LO YM ET AL.: "Rapid clearance of fetal DNA from maternal plasma", AM J HUM GENET, vol. 64, 1999, pages 218 - 24
MARCO AGOSTINI ET AL: "Circulating Cell-Free DNA: A Promising Marker of Pathologic Tumor Response in Rectal Cancer Patients Receiving Preoperative Chemoradiotherapy", ANNALS OF SURGICAL ONCOLOGY, SPRINGER-VERLAG, NE, vol. 18, no. 9, 17 March 2011 (2011-03-17), pages 2461 - 2468, XP019945160, ISSN: 1534-4681, DOI: 10.1245/S10434-011-1638-Y *
MARGULIES M ET AL.: "Genome sequencing in microfabricated high-density picolitre reactors", NATURE, vol. 437, 31 July 2005 (2005-07-31), pages 376 - 80
MCBRIDE DJ ET AL.: "Use of cancer-specific genomic rearrangements to quantify disease burden in plasma from patients with solid tumors", GENES CHROMOSOMES CANCER, vol. 49, 2010, pages 1062 - 9, XP055064914, DOI: doi:10.1002/gcc.20815
METZKER ML: "Sequencing technologies - the next generation", NATURE REVIEWS GENETICS, vol. 11, 2010, pages 31
MIILLER I ET AL.: "Identification of loss of heterozygosity on circulating free DNA in peripheral blood of prostate cancer patients: potential and technical improvements", CLIN CHEM, vol. 54, 15 February 2008 (2008-02-15), pages 688 - 96, XP008095938, DOI: doi:10.1373/clinchem.2007.099333
NAGRATH S ET AL.: "Isolation of rare circulating tumour cells in cancer patients by microchip technology", NATURE, vol. 450, 2007, pages 1235 - 9, XP002681217
NAWROZ H ET AL.: "Microsatellite alterations in serum DNA of head and neck cancer patients", NAT MED, vol. 2, 1996, pages 1035 - 7, XP008124609, DOI: doi:10.1038/nm0996-1035
OLDENHUIS C.N.A.M.: "Prognostic versus predictive value of biomarkers in oncology", EUROPEAN JOURNAL OF CANCER, vol. 44, 2008, pages 946 - 953, XP022625020, DOI: doi:10.1016/j.ejca.2008.03.006
PANTEL K ET AL.: "Detection, clinical relevance and specific biological properties of disseminating tumour cells", NAT REV CANCER, vol. 8, 2008, pages 329 - 40
PAPAGEORGIOU EA ET AL.: "Fetal-specific DNA methylation ratio permits noninvasive prenatal diagnosis of trisomy 21", NAT MED, vol. 17, 6 March 2011 (2011-03-06), pages 510 - 3, XP055358566, DOI: doi:10.1038/nm.2312
PINZANI P ET AL.: "Circulating nucleic acids in cancer and pregnancy", METHODS, vol. 50, 8 February 2010 (2010-02-08), pages 302 - 7, XP026941724, DOI: doi:10.1016/j.ymeth.2010.02.004
RIETHDORF S ET AL.: "Detection of circulating tumor cells in peripheral blood of patients with metastatic breast cancer: a validation study of the CellSearch System", CLIN CANCER RES, vol. 13, 2007, pages 920 - 928, XP002516029, DOI: doi:10.1158/1078-0432.CCR-06-1695
SCHWARZENBACH H ET AL.: "Cell-free nucleic acids as biomarkers in cancer patients", NAT REV CANCER, vol. 11, 12 May 2011 (2011-05-12), pages 426 - 37, XP055247315, DOI: doi:10.1038/nrc3066
SCHWARZENBACH H ET AL.: "Cell-free tumor DNA in blood plasma as a marker for circulating tumor cells in prostate cancer", CLIN CANCER RES, vol. 15, 2009, pages 1032 - 8, XP055083221, DOI: doi:10.1158/1078-0432.CCR-08-1910
See also references of EP2771483A1 *
SIDRANSKY D: "Emerging molecular markers of cancer", NAT REV CANCER, vol. 2, 2002, pages 210 - 9
STROUN M ET AL.: "Neoplastic characteristics of the DNA found in the plasma of cancer patients", ONCOLOGY, vol. 46, 1989, pages 318 - 22
UMETANI N ET AL.: "Prediction of breast tumor progression by integrity of free circulating DNA in serum", J CLIN ONCOL, vol. 24, 2006, pages 4270 - 6, XP002469075, DOI: doi:10.1200/JCO.2006.05.9493
VAN DER VAART M; PRETORIUS PJ: "Is the role of circulating DNA as a biomarker of cancer being prematurely overrated?", CLIN BIOCHEM, vol. 43, 9 September 2009 (2009-09-09), pages 26 - 36, XP026821783
WALTHER A ET AL.: "Genetic prognostic and predictive markers in colorectal cancer", NAT REV CANCER, vol. 9, 18 June 2009 (2009-06-18), pages 489 - 99, XP002567046, DOI: doi:10.1038/nrc2645
WANG BG ET AL.: "Increased plasma DNA integrity in cancer patients", CANCER RES, vol. 63, pages 3966 - 8, XP002510473
WOOD LD ET AL.: "The genomic landscapes of human breast and colorectal cancers", SCIENCE, vol. 318, 11 October 2007 (2007-10-11), pages 1108 - 13, XP055017777, DOI: doi:10.1126/science.1145720
YACHIDA S ET AL.: "Distant metastasis occurs late during the genetic evolution of pancreatic cancer", NATURE, vol. 467, 2010, pages 1114 - 7
YUNG TK ET AL.: "Single-molecule detection of epidermal growth factor receptor mutations in plasma by microfluidics digital PCR in non-small cell lung cancer patients", CLIN CANCER RES, vol. 15, 10 March 2009 (2009-03-10), pages 2076 - 84, XP002628813, DOI: doi:10.1158/1078-0432.CCR-08-2622

Cited By (40)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US11217330B2 (en) 2012-03-08 2022-01-04 The Chinese University Of Hong Kong Size-based analysis of fetal DNA fraction in plasma
US11031100B2 (en) 2012-03-08 2021-06-08 The Chinese University Of Hong Kong Size-based sequencing analysis of cell-free tumor DNA for classifying level of cancer
US10741270B2 (en) 2012-03-08 2020-08-11 The Chinese University Of Hong Kong Size-based analysis of cell-free tumor DNA for classifying level of cancer
EP3047038A4 (fr) * 2013-09-20 2017-04-05 The Chinese University Of Hong Kong Analyse par séquençage de l'adn circulant en vue de la détection et du suivi de maladies auto-immunes
US10174375B2 (en) 2013-09-20 2019-01-08 The Chinese University Of Hong Kong Sequencing analysis of circulating DNA to detect and monitor autoimmune diseases
EP3047038A1 (fr) * 2013-09-20 2016-07-27 The Chinese University Of Hong Kong Analyse par séquençage de l'adn circulant en vue de la détection et du suivi de maladies auto-immunes
EP3617323A1 (fr) * 2013-09-20 2020-03-04 The Chinese University Of Hong Kong Analyse d'adn circulant pour détecter et surveiller la maladie auto-immune du lupus érythémateux systémique
WO2016008550A1 (fr) 2014-07-16 2016-01-21 Roche Diagnostics Gmbh Détection précoce non invasive de rejet de greffe d'organe solide par analyse quantitative d'amplicons du gène hla
US11984195B2 (en) 2014-07-18 2024-05-14 The Chinese University Of Hong Kong Methylation pattern analysis of tissues in a DNA mixture
US11062789B2 (en) 2014-07-18 2021-07-13 The Chinese University Of Hong Kong Methylation pattern analysis of tissues in a DNA mixture
US11352670B2 (en) 2014-07-25 2022-06-07 University Of Washington Methods of determining tissues and/or cell types giving rise to cell-free DNA, and methods of identifying a disease or disorder using same
CN107407626A (zh) * 2014-09-26 2017-11-28 加利福尼亚大学董事会 评估癌症的疾病状况的方法
US10364467B2 (en) 2015-01-13 2019-07-30 The Chinese University Of Hong Kong Using size and number aberrations in plasma DNA for detecting cancer
US11581063B2 (en) 2015-07-23 2023-02-14 The Chinese University Of Hong Kong Analysis of fragmentation patterns of cell-free DNA
US11615865B2 (en) 2015-07-23 2023-03-28 The Chinese University Of Hong Kong Analysis of fragmentation patterns of cell-free DNA
US10453556B2 (en) 2015-07-23 2019-10-22 The Chinese University Of Hong Kong Analysis of fragmentation patterns of cell-free DNA
US11605445B2 (en) 2015-07-23 2023-03-14 The Chinese University Of Hong Kong Analysis of fragmentation patterns of cell-free DNA
US11534756B2 (en) 2016-03-24 2022-12-27 Biological Dynamics, Inc. Compact device for detection of nanoscale analytes
US11062791B2 (en) 2016-09-30 2021-07-13 Guardant Health, Inc. Methods for multi-resolution analysis of cell-free nucleic acids
US9850523B1 (en) 2016-09-30 2017-12-26 Guardant Health, Inc. Methods for multi-resolution analysis of cell-free nucleic acids
US11817179B2 (en) 2016-09-30 2023-11-14 Guardant Health, Inc. Methods for multi-resolution analysis of cell-free nucleic acids
US11817177B2 (en) 2016-09-30 2023-11-14 Guardant Health, Inc. Methods for multi-resolution analysis of cell-free nucleic acids
US11459616B2 (en) 2016-10-24 2022-10-04 The Chinese University Of Hong Kong Methods and systems for tumor detection
US11435339B2 (en) 2016-11-30 2022-09-06 The Chinese University Of Hong Kong Analysis of cell-free DNA in urine
US11479825B2 (en) 2017-01-25 2022-10-25 The Chinese University Of Hong Kong Diagnostic applications using nucleic acid fragments
US10633713B2 (en) 2017-01-25 2020-04-28 The Chinese University Of Hong Kong Diagnostic applications using nucleic acid fragments
EP3728582A4 (fr) * 2017-12-19 2021-09-01 Biological Dynamics, Inc. Procédés et dispositifs de détection et de quantification de fragments d'adn acellulaire
US11731132B2 (en) 2017-12-19 2023-08-22 Biological Dynamics, Inc. Methods and devices for detection of multiple analytes from a biological sample
JP2021509266A (ja) * 2017-12-19 2021-03-25 バイオロジカル ダイナミクス，インク． 無細胞ｄｎａ断片を検出および定量化するための方法とデバイス
US11883833B2 (en) 2018-04-02 2024-01-30 Biological Dynamics, Inc. Dielectric materials
US11410750B2 (en) 2018-09-27 2022-08-09 Grail, Llc Methylation markers and targeted methylation probe panel
US11685958B2 (en) 2018-09-27 2023-06-27 Grail, Llc Methylation markers and targeted methylation probe panel
US11725251B2 (en) 2018-09-27 2023-08-15 Grail, Llc Methylation markers and targeted methylation probe panel
US11795513B2 (en) 2018-09-27 2023-10-24 Grail, Llc Methylation markers and targeted methylation probe panel
US11643693B2 (en) 2019-01-31 2023-05-09 Guardant Health, Inc. Compositions and methods for isolating cell-free DNA
WO2020190613A1 (fr) * 2019-03-15 2020-09-24 Stitch Bio, Llc Procédés de détection d'une maladie résiduelle minimale
WO2021023650A1 (fr) * 2019-08-02 2021-02-11 INSERM (Institut National de la Santé et de la Recherche Médicale) Procedes de depistage d'un cancer chez un sujet
US11211144B2 (en) 2020-02-18 2021-12-28 Tempus Labs, Inc. Methods and systems for refining copy number variation in a liquid biopsy assay
US11211147B2 (en) 2020-02-18 2021-12-28 Tempus Labs, Inc. Estimation of circulating tumor fraction using off-target reads of targeted-panel sequencing
US11475981B2 (en) 2020-02-18 2022-10-18 Tempus Labs, Inc. Methods and systems for dynamic variant thresholding in a liquid biopsy assay

Also Published As

Publication number Publication date
EP2771483A1 (fr) 2014-09-03

Similar Documents

Publication Publication Date Title
EP2771483A1 (fr) Méthode pour diagnostiquer une maladie basée sur la distribution de l'adn plasmatique
JP6683752B2 (ja) 血漿による胎児または腫瘍のメチロームの非侵襲的決定
TWI730973B (zh) 游離ｄｎａ(ｃｅｌｌ-ｆｒｅｅ ｄｎａ)之片段化模式分析
KR102529113B1 (ko) 소변 및 기타 샘플에서의 무세포 dna의 분석
EP3543356B1 (fr) Analyse de motifs de méthylation de tissus dans un mélange d'adn
DK2860266T3 (en) Size-based analysis of DNA to classify a cancer level
JP2022519159A (ja) 循環細胞の分析方法
Dietz et al. Low input whole-exome sequencing to determine the representation of the tumor exome in circulating DNA of non-small cell lung cancer patients
US20190341127A1 (en) Size-tagged preferred ends and orientation-aware analysis for measuring properties of cell-free mixtures
WO2020192680A1 (fr) Détermination de formes linéaires et circulaires d'acides nucléiques circulants
Udomruk et al. Size distribution of cell-free DNA in oncology
Cochran et al. Analysis of BRCA2 loss of heterozygosity in tumor tissue using droplet digital polymerase chain reaction
KR102236717B1 (ko) 조혈모세포 이식 후 혈액암 예후 예측을 위한 정보 제공 방법
Piérard et al. Streamlining molecular pathobiology of malignant melanoma
Taylor Biomarkers of Lung Cancer Risk and Progression
An The Current State of Molecular Pathology in Diagnosing Sarcomas
BR122021021820B1 (pt) Método para determinar um perfil de metilação de uma amostra biológica de um organismo e meio de armazenamento legível por computador

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 12780167

Country of ref document: EP

Kind code of ref document: A1

WWE Wipo information: entry into national phase

Ref document number: 2012780167

Country of ref document: EP

NENP Non-entry into the national phase

Ref country code: DE