WO2005043162A2 - Method for distinguishing mll-ptd-positive aml from other aml subtypes - Google Patents

Method for distinguishing mll-ptd-positive aml from other aml subtypes Download PDF

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WO2005043162A2
WO2005043162A2 PCT/EP2004/012464 EP2004012464W WO2005043162A2 WO 2005043162 A2 WO2005043162 A2 WO 2005043162A2 EP 2004012464 W EP2004012464 W EP 2004012464W WO 2005043162 A2 WO2005043162 A2 WO 2005043162A2
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Prior art keywords
numbers
expression
polynucleotide
ptd
polynucleotide defined
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PCT/EP2004/012464
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French (fr)
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WO2005043162A8 (en
WO2005043162A3 (en
Inventor
Torsten Haferlach
Martin Dugas
Wolfgang Kern
Alexander Kohlmann
Susanne Schnittger
Claudia Schoch
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Roche Diagnostics Gmbh
F.Hoffmann-La Roche Ag
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Priority to US10/576,093 priority Critical patent/US20070212687A1/en
Priority to EP04797591A priority patent/EP1682902A2/en
Publication of WO2005043162A2 publication Critical patent/WO2005043162A2/en
Publication of WO2005043162A8 publication Critical patent/WO2005043162A8/en
Publication of WO2005043162A3 publication Critical patent/WO2005043162A3/en

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57407—Specifically defined cancers
    • G01N33/57426—Specifically defined cancers leukemia
    • 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
    • C12Q2600/158—Expression markers

Definitions

  • the present invention is directed to a method for distinguishing MLL-PTD-positive AML from other AML subtypes by determining the expression level of selected marker genes.
  • Leukemias are classified into four different groups or types: acute myeloid (AML), acute lymphatic (ALL), chronic myeloid (CML) and chronic lymphatic leukemia (CLL). Within these groups, several subcategories can be identified further using a panel of standard techniques as described below. These different subcategories in leukemias are associated with varying clinical outcome and therefore are the basis for different treatment strategies. The importance of highly specific classification may be illustrated in detail further for the AML as a very heterogeneous group of diseases. Effort is aimed at identifying biological entities and to distinguish and classify subgroups of AML which are associated with a favorable, intermediate or unfavorable prognosis, respectively.
  • the FAB classification was proposed by the French- American-British co-operative group which was based on cytomorphology and cytochemistry in order to separate AML subgroups according to the morphological appearance of blasts in the blood and bone marrow.
  • genetic abnormalities occurring in the leukemic blast had a major impact on the morphological picture and even more on the prognosis.
  • the karyotype of the leukemic blasts is the most important independent prognostic factor regarding response to therapy as well as survival.
  • leukemia diagnostics Analysis of the morphology and cytochemistry of bone marrow blasts and peripheral blood cells is necessary to establish the diagnosis. In some cases the addition of immunophenotyping is mandatory to separate very undifferentiated AML from acute lymphoblastic leukemia and CLL. Leukemia subtypes investigated can be diagnosed by cytomorphology alone, only if an expert reviews the smears. However, a genetic analysis based on chromosome analysis, fluorescence in situ hybridization or RT-
  • PCR and immunophenotyping is required in order to assign all cases in to the right category.
  • the aim of these techniques besides diagnosis is mainly to determine the prognosis of the leukemia.
  • a major disadvantage of these methods, however, is that viable cells are necessary as the cells for genetic analysis have to divide in vitro in order to obtain metaphases for the analysis.
  • Another problem is the long time of 72 hours from receipt of the material in the laboratory to obtain the result.
  • great experience in preparation of chromosomes and even more in analyzing the karyotypes is required to obtain the correct result in at least 90% of cases.
  • CML chronic myeloid leukemia
  • CLL chronic lymphatic
  • ALL acute lymphoblastic
  • AML acute myeloid leukemia
  • the new therapeutic drug inhibits the CML specific chimeric tyrosine kinase BCR-ABL generated from the genetic defect observed in
  • the technical problem underlying the present invention was to provide means for leukemia diagnostics which overcome at least some of the disadvantages of the prior art diagnostic methods, in particular encompassing the time-consuming and unreliable combination of different methods and which provides a rapid assay to unambiguously distinguish one AML subtype from another, e.g. by genetic analysis.
  • WO-A 03/039443 discloses marker genes the expression levels of which are characteristic for certain leukemia, e.g. AML subtypes and additionally discloses methods for differentiating between the subtype of AML cells by determining the expression profile of the disclosed marker genes.
  • WO-A 03/039443 does not provide guidance which set of distinct genes discriminate between two subtypes and, as such, can be routineously taken in order to distinguish one AML subtype from another.
  • the problem is solved by the present invention, which provides a method for distinguishing MLL-PTD-positive AML from other AML subtypes in a sample, the method comprising determining the expression level of markers selected from the markers identifiable by their Affymetrix Identification Numbers (affy id) as defined in Tables 1, 2, and/or 3, wherein a lower expression of at least one polynucleotide defined by any of the numbers 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, and/or 50 of Table 1 is indicative for the presence of PTD (MLL-PTD-positive AML with normal karyotype) when PTD is distinguished from AML_NK (MLL- PTD-negative AML with normal karyotype), and/or wherein a lower expression of at least one polynucleotide defined by
  • tMLL AML with translocations in the MLL gene (t(l Iq23)/MLL)
  • PTD AML with normal karyotype and Partial Tandem Duplication (PTD) within the MLL gene (MLL-PTD)
  • AMLJSfK AML with normal karyotype (no Partial Tandem Duplication (PTD) within the MLL gene) t(8;21)
  • all other subtypes refer to the subtypes of the present invention, i.e. if one subtype is distinguished from “all other subtypes", it is distiguished from all other subtypes contained in the present invention.
  • a sample means any biological material containing genetic information in the form of nucleic acids or proteins obtainable or obtained from an individual.
  • the sample includes e.g. tissue samples, cell samples, bone marrow and/or body fluids such as blood, saliva, semen.
  • the sample is blood or bone marrow, more preferably the sample is bone marrow.
  • a general method for isolating and preparing nucleic acids from a sample is outlined in Example 3.
  • the term "lower expression” is generally assigned to all by numbers and Affymetrix Id. definable polynucleotides the t- values and fold change (fc) values of which are negative, as indicated in the Tables. Accordingly, the term “higher expression” is generally assigned to all by numbers and Affymetrix Id. definable polynucleotides the t-values and fold change (fc) values of which are positive.
  • the term "expression” refers to the process by which mRNA or a polypeptide is produced based on the nucleic acid sequence of a gene, i.e.illerexpression" also includes the formation of mRNA upon transcription.
  • the term determining the expression level preferably refers to the determination of the level of expression, namely of the markers.
  • markers refers to any genetically controlled difference which can be used in the genetic analysis of a test versus a control sample, for the purpose of assigning the sample to a defined genotype or phenotype.
  • markers refer to genes which are differentially expressed in, e.g., different AML subtypes.
  • the markers can be defined by their gene symbol name, their encoded protein name, their transcript identification number (cluster identification number), the data base accession number, public accession number or GenBank identifier or, as done in the present invention, Affymetrix identification number, chromosomal location, UniGene accession number and cluster type, LocusLink accession number (see Examples and Tables).
  • the Affymetrix identification number (affy id) is accessible for anyone and the person skilled in the art by entering the "gene expression omnibus" internet page of the National Center for Biotechnology Information (NCBI) (http://www.ncbi.nlm.nih.gov/geo/).
  • NCBI National Center for Biotechnology Information
  • the affy id's of the polynucleotides used for the method of the present invention are derived from the so-called U133 chip.
  • the expression level of a marker is determined by the determining the expression of its corresponding "polynucleotide" as described hereinafter.
  • the term “bigpolynucleotide” refers, generally, to a DNA, in particular cDNA, or RNA, in particular a cRNA, or a portion thereof or a polypeptide or a portion thereof.
  • the polynucleotide is formed upon transcription of a nucleotide sequence which is capable of expression.
  • the polynucleotide fragments refer to fragments preferably of between at least 8, such as 10, 12, 15 or 18 nucleotides and at least 50, such as 60, 80, 100, 200 or 300 nucleotides in length, or a complementary sequence thereto, representing a consecutive stretch of nucleotides of a gene, cDNA or mRNA.
  • polynucleotides include also any fragment (or complementary sequence thereto) of a sequence derived from any of the markers defined above as long as these fragments unambiguously identify the marker.
  • the determination of the expression level may be effected at the transcriptional or translational level, i.e. at the level of mRNA or at the protein level.
  • Protein fragments such as peptides or polypeptides advantageously comprise between at least 6 and at least 25, such as 30, 40, 80, 100 or 200 consecutive amino acids representative of the corresponding full length protein. Six amino acids are generally recognized as the lowest peptidic stretch giving rise to a linear epitope recognized by an antibody, fragment or derivative thereof.
  • the proteins or fragments thereof may be analysed using nucleic acid molecules specifically binding to three-dimensional structures (aptamers).
  • the determination of the expression levels may be effected by a variety of methods.
  • the polynucleotide, in particular the cRNA is labelled.
  • the labelling of the polynucleotide or a polypeptide can occur by a variety of methods known to the skilled artisan.
  • the label can be fluorescent, chemiluminescent, bioluminescent, radioactive (such as 3 H or 32 P).
  • the labelling compound can be any labelling compound being suitable for the labelling of polynucleotides and/or polypeptides. Examples include fluorescent dyes, such as fluorescein, dichlorofluorescein, hexachlorofluorescein, BODIPY variants, ROX, tetramethylrhodamin, rhodamin X, Cyanine-2, Cyanine-3, Cyanine-5, Cyanine-7,
  • IRD40 FluorX, Oregon Green, Alexa variants (available e.g. from Molecular Probes or Amersham Biosciences) and the like, biotin or biotinylated nucleotides, digoxigenin, radioisotopes, antibodies, enzymes and receptors.
  • the detection is done via fluorescence measurements, conjugation to streptavidin and/or avidin, antigen-antibody- and/or antibody- ntibody- interactions, radioactivity measurements, as well as catalytic and or receptor/ligand interactions.
  • Suitable methods include the direct labelling (incorporation) method, the amino-modified (amino-allyl) nucleotide method (available e.g.
  • DNA dendrimer labelling as kit available e.g. from Genisphere
  • biotin or biotinylated nucleotides for labelling are directly incorporated into, e.g. the cRNA polynucleotide by in vitro transcription.
  • cDNA may be prepared into which a detectable label, as exemplified above, is incorporated. Said detectably labelled cDNA, in single-stranded form, may then be hybridised, preferably under stringent or highly stringent conditions to a panel of single-stranded oligonucleotides representing different genes and affixed to a solid support such as a chip. Upon applying appropriate washing steps, those cDNAs will be detected or quantitatively detected that have a counterpart in the oligonucleotide panel.
  • the mRNA or the cDNA may be amplified e.g.
  • the cDNAs are transcribed into cRNAs prior to the hybridisation step wherein only in the transcription step a label is incorporated into the nucleic acid and wherein the cRNA is employed for hybridisation.
  • the label may be attached subsequent to the transcription step.
  • proteins from a cell or tissue under investigation may be contacted with a panel of aptamers or of antibodies or fragments or derivatives thereof.
  • the antibodies etc. may be affixed to a solid support such as a chip. Binding of proteins indicative of an AML subtype may be verified by binding to a detectably labelled secondary antibody or aptamer.
  • a detectably labelled secondary antibody or aptamer For the labelling of antibodies, it is referred to
  • a minimum set of proteins necessary for diagnosis of all AML subtypes may be selected for creation of a protein array system to make diagnosis on a protein lysate of a diagnostic bone marrow sample directly.
  • Protein Array Systems for the detection of specific protein expression profiles already are available (for example: Bio-Plex, BIORAD, Munchen, Germany).
  • antibodies against the proteins have to be produced and immobilized on a platform e.g. glasslides or microtiterplates.
  • the immobilized antibodies can be labelled with a reactant specific for the certain target proteins as discussed above.
  • the reactants can include enzyme substrates, DNA, receptors, antigens or antibodies to create for example a capture sandwich irnmunoassay.
  • the expression of more than one of the above defined markers is determined.
  • the statistical significance of markers as expressed in q or p values based on the concept of the false discovery rate is determined. In doing so, a measure of statistical significance called the q value is associated with each tested feature.
  • the q value is similar to the p value, except it is a measure of significance in terms of the false discovery rate rather than the false positive rate (Storey JD and Tibsbirani R. Proc.Natl.Acad.Sci., 2003, Vol. 100:9440-5.
  • markers as defined in Table 1.1-3.15 having a q- value of less than 3E-03, more preferred less than 1.5E-09, most preferred less than 1.5E-11, less than 1.5E-20, less than 1.5E-30, are measured.
  • the expression level of at least 2, of at least 5, of at least 10 out of the markers having the numbers 1 - 10, 1-20, 1-40, 1-50 of at least one of the Tables are measured.
  • the level of the expression of the handheldmarker i.e. the expression of the polynucleotide is indicative of the AML subtype of a cell or an organism.
  • the level of expression of a marker or group of markers is measured and is compared with the level of expression of the same marker or the same group of markers from other cells or samples. The comparison may be effected in an actual experiment or in silico.
  • expression level also referred to as expression pattern or expression signature (expression profile)
  • the difference at least is 5 %, 10% or 20%, more preferred at least 50% or may even be as high as 75% or 100%. More prefened the difference in the level of expression is at least 200%, i.e. two fold, at least 500%, i.e. five fold, or at least 1000%, i.e. 10 fold.
  • the expression level of markers expressed lower in a first subtype than in at least one second subtype, which differs from the first subtype is at least
  • the expression level of markers expressed higher in a first subtype than in at least one second subtype, which differs from the first subtype is at least 5 %, 10% or 20%, more preferred at least 50% or may even be 75% or 100%, i.e. 2-fold higher, preferably at least 10-fold, more preferably at least 50-fold, and most preferably at least 100-fold lower in the first subtype.
  • the expression level of markers expressed higher in a first subtype than in at least one second subtype, which differs from the first subtype is at least 5 %, 10% or 20%, more preferred at least 50% or may even be 75% or 100%, i.e. 2-fold higher, preferably at least 10-fold, more preferably at least 50-fold, and most preferably at least 100-fold higher in the first subtype.
  • the sample is derived from an individual having leukaemia, preferably AML.
  • the polynucleotide the expression level of which is determined is in form of a transcribed polynucleotide.
  • a particularly preferred transcribed polynucleotide is an mRNA, a cDNA and/or a cRNA, with the latter being preferred.
  • Transcribed polynucleotides are isolated from a sample, reverse transcribed and/or amplified, and labelled, by employing methods well-known the person skilled in the art (see Example 3).
  • the step of determining the expression profile further comprises amplifying the transcribed polynucleotide.
  • the method comprises hybridizing the transcribed polynucleotide to a complementary polynucleotide, or a portion thereof, under stringent hybridization conditions, as described hereinafter.
  • hybridizing means hybridization under conventional hybridization conditions, preferably under stringent conditions as described, for example, in Sambrook, J., et al., in "Molecular Cloning: A Laboratory Manual” (1989), Eds. J. Sambrook, E. F. Fritsch and T. Maniatis, Cold Spring Harbour Laboratory Press,
  • Such conditions are, for example, hybridization in 6x SSC, pH 7.0 / 0.1% SDS at about 45°C for 18-23 hours, followed by a washing step with 2x SSC/0.1% SDS at 50°C.
  • the salt concentration in the washing step can for example be chosen between 2x SSC/0.1% SDS at room temperature for low stringency and 0.2x SSC/0.1% SDS at 50°C for high stringency.
  • the temperature of the washing step can be varied between room temperature, ca. 22°C, for low stringency, and 65°C to 70° C for high stringency.
  • polynucleotides that hybridize at lower stringency hybridization conditions. Changes in the stringency of hybridization and signal detection are primarily accomplished through the manipulation, preferably of formamide concentration
  • washes performed following stringent hybridization can be done at higher salt concentrations (e.g. 5x SSC).
  • Variations in the above conditions may be accomplished through the inclusion and/or substitution of alternate blocking reagents used to suppress background in hybridization experiments.
  • the inclusion of specific blocking reagents may require modification of the hybridization conditions described above, due to problems with compatibility.
  • “Complementary” and “complementarity”, respectively, can be described by the percentage, i.e. proportion, of nucleotides which can form base pairs between two polynucleotide strands or within a specific region or domain of the two strands.
  • complementary nucleotides are, according to the base pairing rules, adenine and thymine (or adenine and uracil), and cytosine and guanine.
  • Complementarity may be partial, in which only some of the nucleic acids' bases are matched according to the base pairing rules. Or, there may be a complete or total complementarity between the nucleic acids. The degree of complementarity between nucleic acid strands has effects on the efficiency and strength of hybridization between nucleic acid strands.
  • Two nucleic acid strands are considered to be 100% complementary to each other over a defined length if in a defined region all adenines of a first strand can pair with a thymine (or an uracil) of a second strand, all guanines of a first strand can pair with a cytosine of a second strand, all thymine (or uracils) of a first strand can pair with an adenine of a second strand, and all cytosines of a first strand can pair with a guanine of a second strand, and vice versa.
  • the degree of complementarity is determined over a stretch of 20, preferably 25, nucleotides, i.e.
  • a 60% complementarity means that within a region of 20 nucleotides of two nucleic acid strands 12 nucleotides of the first strand can base pair with 12 nucleotides of the second strand according to the above ruling, either as a stretch of 12 contiguous nucleotides or interspersed by non-pairing nucleotides, when the two strands are attached to each other over said region of 20 nucleotides.
  • the degree of complementarity can range from at least about 50% to full, i.e. 100% complementarity.
  • Two single nucleic acid strands are said to be "substantially complementary" when they are at least about 80% complementary, preferably about 90% or higher. For carrying out the method of the present invention substantial complementarity is preferred.
  • Prefened methods for detection and quantification of the amount of polynucleotides i.e. for the methods according to the invention allowing the determination of the level of expression of a marker, are those described by Sambrook et al. (1989) or real time methods known in the art as the TaqMan® method disclosed in WO92/02638 and the corresponding U.S. 5,210,015, U.S. 5,804,375, U.S. 5,487,972. This method exploits the exonuclease activity of a polymerase to generate a signal.
  • the (at least one) target nucleic acid component is detected by a process comprising contacting the sample with an oligonucleotide containing a sequence complementary to a region of the target nucleic acid component and a labeled oligonucleotide containing a sequence complementary to a second region of the same target nucleic acid component sequence strand, but not including the nucleic acid sequence defined by the first oligonucleotide, to create a mixture of duplexes during hybridization conditions, wherein the duplexes comprise the target nucleic acid annealed to the first oligonucleotide and to the labeled oligonucleotide such that the 3 '-end of the first oligonucleotide is adjacent to the 5 '-end of the labeled oligonucleotide.
  • this mixture is treated with a template-dependent nucleic acid polymerase having a 5' to 3' nuclease activity under conditions sufficient to permit the 5' to 3' nuclease activity of the polymerase to cleave the annealed, labeled oligonucleotide and release labeled fragments.
  • the signal generated by the hydrolysis of the labeled oligonucleotide is detected and/ or measured.
  • TaqMan® technology eliminates the need for a solid phase bound reaction complex to be formed and made detectable.
  • Other methods include e.g. fluorescence resonance energy transfer between two adjacently hybridized probes as used in the LightCycler® format described in U.S. 6,174,670.
  • Example 3 A prefened protocol if the marker, i.e. the polynucleotide, is in form of a transcribed nucleotide, is described in Example 3, where total RNA is isolated, cDNA and, subsequently, cRNA is synthesized and biotin is incorporated during the transcription reaction.
  • the purified cRNA is applied to commercially available arcays which can be obtained e.g. from Affymetrix.
  • the hybridized cRNA is detected according to the methods described in Example 3.
  • the anays are produced by photolithography or other methods known to experts skilled in the art e.g. from U.S. 5,445,934, U.S. 5,744,305, U.S. 5,700,637, U.S. 5,945,334 and EP 0 619 321 or EP 0373 203, or as decribed hereinafter in greater detail.
  • the polynucleotide or at least one of the polynucleotides is in form of a polypeptide.
  • the expression level of the polynucleotides or polypeptides is detected using a compound which specifically binds to the polynucleotide of the polypeptide of the present invention.
  • binding means that the compound is capable of discriminating between two or more polynucleotides or polypeptides, i.e. it binds to the desired polynucleotide or polypeptide, but essentially does not bind unspecifically to a different polynucleotide or polypeptide.
  • the compound can be an antibody, or a fragment thereof, an enzyme, a so-called small molecule compound, a protein-scaffold, preferably an anticalin.
  • the compound specifically binding to the polynucleotide or polypeptide is an antibody, or a fragment thereof.
  • an "antibody” comprises monoclonal antibodies as first described by Kohler and Milstein in Nature 278 (1975), 495-497 as well as polyclonal antibodies, i.e. entibodies contained in a polyclonal antiserum.
  • Monoclonal antibodies include those produced by transgenic mice. Fragments of antibodies include F(ab') 2 , Fab and Fv fragments. Derivatives of antibodies include scFvs, chimeric and humanized antibodies. See, for example Harlow and Lane, loc. cit.
  • the person skilled in the art is aware of a variety of methods, all of which are included in the present invention.
  • Examples include immunoprecipitation, Western blotting, Enzyme-linked immuno sorbent assay (ELISA), Enzyme-linked imrnuno sorbent assay (RIA), dissociation-enhanced lanthanide fluoro immuno assay (DELFIA), scintillation proximity assay (SPA).
  • ELISA Enzyme-linked immuno sorbent assay
  • RIA Enzyme-linked imrnuno sorbent assay
  • DELFIA dissociation-enhanced lanthanide fluoro immuno assay
  • SPA scintillation proximity assay
  • the method for distinguishing MLL-PTD-positive AML from other AML subtypes is canied out on an anay.
  • an “anay” or “microanay” refers to a linear or two- or three dimensional anangement of preferably discrete nucleic acid or polypeptide probes which comprises an intentionally created collection of nucleic acid or polypeptide probes of any length spotted onto a substrate/solid support.
  • a collection of nucleic acids or polypeptide spotted onto a substrate/solid support also under the term "anay”.
  • a microanay usually refers to a miniaturised anay anangement, with the probes being attached to a density of at least about 10, 20, 50, 100 nucleic acid molecules refening to different or the same genes per cm .
  • an anay can be refened to as "gene chip”.
  • the anay itself can have different formats, e.g. libraries of soluble probes or libraries of probes tethered to resin beads, silica chips, or other solid supports.
  • the process of anay fabrication is well-known to the person skilled in the art.
  • the process for preparing a nucleic acid anay comprises preparing a glass (or other) slide (e.g. chemical treatment of the glass to enhance binding of the nucleic acid probes to the glass surface), obtaining DNA sequences representing genes of a genome of interest, and spotting sequences these sequences of interest onto glass slide.
  • Sequences of interest can be obtained via creating a cDNA library from an mRNA source or by using publicly available databases, such as GeneBank, to annotate the sequence info mation of custom cDNA libraries or to identify cDNA clones from previously prepared libraries.
  • the liquid containing the amplified probes can be deposited on the anay by using a set of microspotting pins. Ideally, the amount deposited should be uniform.
  • the process can further include UV-crosslinking in order to enhance immobilization of the probes on the anay.
  • the anay is a high density oligonucleotide (oligo) anay using a light-directed chemical synthesis process, employing the so-called photolithography technology.
  • oligo anays (according to the Affymetrix technology) use a single-dye technology. Given the sequence information of the markers, the sequence can be synthesized directly onto the anay, thus, bypassing the need for physical intermediates, such as PCR products, required for making cDNA anays.
  • the marker, or partial sequences thereof can be represented by 14 to 20 features, preferably by less than 14 features, more preferably less than 10 features, even more preferably by 6 features or less, with each feature being a short sequence of nucleotides (oligonucleotide), which is a perfect match (PM) to a segment of the respective gene.
  • the PM oligonucleotide are paired with mismatch (MM) oligonucleotides which have a single mismatch at the central base of the nucleotide and are used as "controls".
  • the chip exposure sites are defined by masks and are deprotected by the use of light, followed by a chemical coupling step resulting in the synthesis of one nucleotide. The masking, light deprotection, and coupling process can then be repeated to synthesize the next nucleotide, until the nucleotide chain is of the specified length.
  • the method of the present invention is canied out in a robotics system including robotic plating and a robotic liquid transfer system, e.g. using microfluidics, i.e. channelled structured.
  • a particular prefened method according to the present invention is as follows:
  • RNA preferably mRNA
  • the present invention is directed to the use of at least one marker selected from the markers identifiable by their Affymetrix Identification
  • markers for diagnosis of MLL-PTD-positive AML preferably based on microanay technology, offers the following advantages: (1) more rapid and more precise diagnosis, (2) easy to use in laboratories without specialized experience, (3) abolishes the requirement for analyzing viable cells for chromosome analysis (transport problem), and (4) very experienced hematologists for cytomorphology and cytochemistry, immunophenotyping as well as cytogeneticists and molecularbiologists are no longer required.
  • the present invention refers to a diagnostic kit containing at least one marker selected from the markers identifiable by their Affymetrix Identification Numbers (affy id) as defined in Tables 1, and/or 3 for distinguishing MLL-PTD- positive AML from other AML subtypes, in combination with suitable auxiliaries.
  • suitable auxiliaries include buffers, enzymes, labelling compounds, and the like.
  • the marker contained in the kit is a nucleic acid molecule which is capable of hybridizing to the mRNA conesponding to at least one marker of the present invention.
  • the at least one nucleic acid molecule is attached to a solid support, e.g. a polystyrene microtiter dish, nitrocellulose membrane, glass surface or to non-immobilized particles in solution.
  • the diagnostic kit contains at least one reference for a MLL-PTD-positive AML subtype.
  • the reference can be a sample or a data bank.
  • the present invention is directed to an apparatus for distinguishing MLL-PTD-positive AML from other AML subtypes in a sample, containing a reference data bank obtainable by comprising (a) compiling a gene expression profile of a patient sample by determining the expression level at least one marker selected from the markers identifiable by their Affymetrix Identification Numbers (affy id) as defined in Tables 1, and/or 3, and (b) classifying the gene expression profile by means of a machine learning algorithm.
  • affymetrix Identification Numbers as defined in Tables 1, and/or 3
  • the "machine learning algorithm” is a computational-based prediction methodology, also known to the person skilled in the art as “classifier”, employed for characterizing a gene expression profile.
  • the signals conesponding to a certain expression level which are obtained by the microanay hybridization are subjected to the algorithm in order to classify the expression profile.
  • Supervised learning involves "training” a classifier to recognize the distinctions among classes and then “testing” the accuracy of the classifier on an independent test set. For new, unknown sample the classifier shall predict into which class the sample belongs.
  • the machine learning algorithm is selected from the group consisting of Weighted Voting, K-Nearest Neighbors, Decision Tree Induction, Support Vector Machines (SVM), and Feed-Forward Neural Networks.
  • the machine learning algorithm is Support Vector Machine, such as polynomial kernel and Gaussian Radial Basis Function-kernel SVM models.
  • the classification accuracy of a given gene list for a set of microanay experiments is preferably estimated using Support Vector Machines (SVM), because there is evidence that SVM-based prediction slightly outperforms other classification techniques like k-Nearest Neighbors (k-NN).
  • SVM Support Vector Machines
  • the LIBSVM software package version 2.36 was used (SVM-type: C-SVC, linear kernel (http://www.csie.ntu.edu.tw/ ⁇ cjlin/libsvm/)).
  • SVM-type C-SVC, linear kernel (http://www.csie.ntu.edu.tw/ ⁇ cjlin/libsvm/)).
  • the skilled artisan is furthermore refened to Brown et al., Proc.Natl.Acad.Sci., 2000; 97: 262-267, Furey et al, Bioinformatics. 2000; 16: 906-914, and Vapnik V. Statistical Learning Theory. New
  • the classification accuracy of a given gene list for a set of microanay experiments can be estimated using Support Vector Machines (SVM) as supervised learning technique.
  • SVMs are trained using differentially expressed genes which were identified on a subset of the data and then this trained model is employed to assign new samples to those trained groups from a second and different data set. Differentially expressed genes were identified applying ANOVA and t-test-statistics (Welch t-test). Based on identified distinct gene expression signatures respective training sets consisting of 2/3 of cases and test sets with 1/3 of cases to assess classification accuracies are designated. Assignment of cases to training and test set is randomized and balanced by diagnosis. Based on the training set a Support Vector Machine (SVM) model is built.
  • SVM Support Vector Machine
  • the apparent accuracy i.e. the overall rate of conect predictions of the complete data set was estimated by lOfold cross validation.
  • This means that the data set was divided into 10 approximately equally sized subsets, an SVM-model was trained for 9 subsets and predictions were generated for the remaining subset. This training and prediction process was repeated 10 times to include predictions for each subset. Subsequently the data set was split into a training set, consisting of two thirds of the samples, and a test set with the remaining one third. Apparent accuracy for the training set was estimated by lOfold cross validation (analogous to apparent accuracy for complete set). A SVM-model of the training set was built to predict diagnosis in the independent test set, thereby estimating true accuracy of the prediction model. This prediction approach was applied both for overall classification (multi-class) and binary classification (diagnosis X > yes or no). For the latter, sensitivity and specificity were calculated:
  • the reference data bank is backed up on a computational data memory chip which can be inserted in as well as removed from the apparatus of the present invention, e.g. like an interchangeable module, in order to use another data memory chip containing a different reference data bank.
  • the apparatus of the present invention containing a desired reference data bank can be used in a way such that an unknown sample is, first, subjected to gene expression profiling, e.g. by microanay analysis in a manner as described supra or in the art, and the expression level data obtained by the analysis are, second, fed into the apparatus and compared with the data of the reference data bank obtainable by the above method.
  • the apparatus suitably contains a device for entering the expression level of the data, for example a control panel such as a keyboard.
  • the results, whether and how the data of the unknown sample fit into the reference data bank can be made visible on a provided monitor or display screen and, if desired, printed out on an incorporated of connected printer.
  • the apparatus of the present invention is equipped with particular appliances suitable for detecting and measuring the expression profile data and, subsequently, proceeding with the comparison with the reference data bank.
  • the apparatus of the present invention can contain a gripper arm and/or a tray which takes up the microanay containing the hybridized nucleic acids.
  • the present invention refers to a reference data bank for distinguishing MLL-PTD-positive AML from other AML subtypes in a sample obtainable by comprising (a) compiling a gene expression profile of a patient sample by determining the expression level of at least one marker selected from the markers identifiable by their Affymetrix Identification Numbers (affy id) as defined in Tables 1, and/or 3, and (b) classifying the gene expression profile by means of a machine learning algorithm.
  • affymetrix Identification Numbers affy id
  • the reference data bank is backed up and/or contained in a computational memory data chip.
  • Table 1.1-3.15 show AML subtype analysis of MLL-PTD-positive AML versus other AML subtypes. The analysed markers are ordered according to their q-values, beginning with the lowest q-values.
  • Tables 1.1 to 3.15 are accompanied with explanatory tables (Table 1.1 A to 3.15 A) where the numbering and the
  • Affymetrix Id are further defined by other parameters, e.g. gene bank accession number.
  • MLL-PTD Partial tandem duplication within the MLL-gene
  • MLL-PTD Partial tandem duplication within the MLL-gene
  • t(llq23)/MLL the occurence of MLL-PTD is characterized by an unfavourable prognosis.
  • the pathogenetic mechanisms of the MLL-PTD are poorly understood and downstream genes effected by this molecular abenation are not known.
  • AML we performed global gene expression profiling of 184 AML samples at diagnosis using the U133 set of expression microanays (Affymetrix) with >30,000 human genes represented on both anays.
  • Microanay data was analyzed by pattern recognition algorithms (Principal Component Analysis (PCA), hierarchical clustering), as well as Support Vector Machines
  • SVM classification accuracies
  • PTD+ and low in t(l lq23)/MLL samples were HOXB5, HOXB2, MAN1A1, and ZNF207.
  • PCA and hierarchical cluster visualize that the MLL-PTD samples characterize a homogeneous subgroup within AML with normal karyotype, but do not separate from them.
  • Some of the genes that were highly expressed in AML-NK and low in PTD+ were AAK1, RAB4A, HOXA2, BID.
  • genes that were low in AML-NK and high in PTD+ were, among others, MLL, YY1, and SRP46.
  • the training set comprised 83 AML-NK and 19 PTD+ AML cases, the test set 41 AML-NK and 9 PTD+ AML cases, respectively.
  • the 50 test samples were assigned to the conect group with an accuracy of 88%.
  • the methods section contains both information on statistical analyses used for identification of differentially expressed genes and detailed annotation data of identified microanay probesets.
  • sequence data are omitted due to their large size, and because they do not change, whereas the annotation data are updated periodically, for example new information on chromomal location and functional annotation of the respective gene products. Sequence data are available for download in the NetAffx Download
  • Microanay probesets for example found to be differentially expressed between different types of leukemia samples are further described by additional information.
  • HG-U133 ProbeSet D HG-U133 ProbeSetJD describes the probe set identifier. Examples are:
  • GeneChip probe anay name where the respective probeset is represented. Examples are: Affymetrix Human Genome U133A Anay or
  • the Sequence Type indicates whether the sequence is an Exemplar, Consensus or Control sequence.
  • An Exemplar is a single nucleotide sequence taken directly from a public database. This sequence could be an mRNA or EST.
  • a Consensus sequence is a nucleotide sequence assembled by Affymetrix, based on one or more sequence taken from a public database.
  • the cluster identification number with a sub-cluster identifier appended is the cluster identification number with a sub-cluster identifier appended.
  • Sequence Derived From The accession number of the single sequence, or representative sequence on which the probe set is based. Refer to the "Sequence Source” field to determine the database used.
  • a gene symbol and a short title when one is available. Such symbols are assigned by different organizations for different species.
  • Affymetrix annotational data come from the UniGene record. There is no indication which species-specific databank was used, but some of the possibilities include for example HUGO: The Human Genome Organization.
  • the map location describes the chromosomal location when one is available.
  • Cluster type can be "full length” or “est”, or " — “ if unknown.
  • LocusLink This information represents the LocusLink accession number. Full Length Ref. Sequences:
  • the field contains the ID and description for each entry, and there can be multiple entries per probeSet.
  • Example 3 Sample preparation, processing and data analysis
  • Microanay analyses were performed utilizing the GeneChip ® System (Affymetrix, Santa Clara, USA). Hybridization target preparations were performed according to recommended protocols (Affymetrix Technical Manual). In detail, at time of diagnosis, mononuclear cells were purified by Ficoll-Hypaque density centrifugation. They had been lysed immediately in RLT buffer (Qiagen, Hilden, Germany), frozen, and stored at -80°C from 1 week to 38 months. For gene expression profiling cell lysates of the leukemia samples were thawed, homogenized (QIAshredder, Qiagen), and total RNA was extracted (RNeasy Mini).
  • RNA isolated from 1 x 10 7 cells was used as starting material for cDNA synthesis with oligo [(dT) 24 T7promotor] 65 primer (cDNA Synthesis System, Roche Applied Science, Mannheim, Germany). cDNA products were purified by phenol/chlorophorm/IAA extraction (Ambion, Austin, USA) and acetate/ethanol-precipitated overnight. For detection of the hybridized target nucleic acid biotin-labeled ribonucleotides were incorporated during the following in vitro transcription reaction (Enzo BioAnay HighYield RNA Transcript Labeling Kit, Enzo Diagnostics).
  • Suite software (version 5.0.1) extracted fluorescence signal intensities from each feature on the microanays as detected by confocal laser scanning according to the manufacturer's recommendations.
  • Expression analysis quality assessment parameters included visiual anay inspection of the scanned image for the presence of image artifacts and conect grid alignment for the identification of distinct probe cells as well as both low 375' ratio of housekeeping controls (mean: 1.90 for GAPDH) and high percentage of detection calls (mean: 46.3% present called genes).
  • the 3' to 5' ratio of GAPDH probesets can be used to assess RNA sample and assay quality. Signal values of the 3' probe sets for GAPDH are compared to the Signal values of the conesponding
  • the ratio of the 3' probe set to the 5' probe set is generally no more than 3.0.
  • a high 3' to 5' ratio may indicate degraded RNA or inefficient synthesis of ds cDNA or biotinylated cRNA (GeneChip ® Expression Analysis Technical Manual, www.affymetrix.com).
  • Detection calls are used to determine whether the transcript of a gene is detected (present) or undetected (absent) and were calculated using default parameters of the Microanay Analysis Suite MAS 5.0 software package.
  • Method 2 Bone manow (BM) aspirates are taken at the time of the initial diagnostic biopsy and remaining material is immediately lysed in RLT buffer (Qiagen), frozen and stored at -80 C until preparation for gene expression analysis.
  • RLT buffer Qiagen
  • the targets for GeneChip analysis are prepared according to the cunent Expression Analysis. Briefly, frozen lysates of the leukemia samples are thawed, homogenized
  • RNA extracted RNeasy Mini Kit, Qiagen
  • RNA synthesis Kit Oligo-dT-T7-Promotor Primer
  • the cDNA is purified by phenol-chlorophorm extraction and precipitated with 100% Ethanol over night.
  • biotin-labeled ribonucleotides are incorporated during the in vitro transcription reaction (Enzo® BioAnayTM HighYieldTM RNA Transcript Labeling Kit, ENZO).
  • the Affymetrix software (Microanay Suite, Version 4.0.1) extracted fluorescence intensities from each element on the anays as detected by confocal laser scanning according to the manufacturers recommendations .
  • OVA One-Versus-AII
  • _x_at HINT1 1.40 4.69E-12 2.81 E-08 1.68 10.32 5q31.2 19200986.at SERPING1 9.53 1.51 E-09 1.26E-06 1.97 10.29 11q12- q13.1 20201137.

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Abstract

Disclosed is a method for distinguishing MLL-PTD-positive AML from other AML subtypes in a sample by determining the expression level of markers, as well as a diagnostic kit and an apparatus containing the markers.

Description

Method for distinguishing MLL-PTD-positive AML from other AML subtypes
The present invention is directed to a method for distinguishing MLL-PTD-positive AML from other AML subtypes by determining the expression level of selected marker genes.
Leukemias are classified into four different groups or types: acute myeloid (AML), acute lymphatic (ALL), chronic myeloid (CML) and chronic lymphatic leukemia (CLL). Within these groups, several subcategories can be identified further using a panel of standard techniques as described below. These different subcategories in leukemias are associated with varying clinical outcome and therefore are the basis for different treatment strategies. The importance of highly specific classification may be illustrated in detail further for the AML as a very heterogeneous group of diseases. Effort is aimed at identifying biological entities and to distinguish and classify subgroups of AML which are associated with a favorable, intermediate or unfavorable prognosis, respectively. In 1976, the FAB classification was proposed by the French- American-British co-operative group which was based on cytomorphology and cytochemistry in order to separate AML subgroups according to the morphological appearance of blasts in the blood and bone marrow. In addition, it was recognized that genetic abnormalities occurring in the leukemic blast had a major impact on the morphological picture and even more on the prognosis. So far, the karyotype of the leukemic blasts is the most important independent prognostic factor regarding response to therapy as well as survival.
Usually, a combination of methods is necessary to obtain the most important information in leukemia diagnostics: Analysis of the morphology and cytochemistry of bone marrow blasts and peripheral blood cells is necessary to establish the diagnosis. In some cases the addition of immunophenotyping is mandatory to separate very undifferentiated AML from acute lymphoblastic leukemia and CLL. Leukemia subtypes investigated can be diagnosed by cytomorphology alone, only if an expert reviews the smears. However, a genetic analysis based on chromosome analysis, fluorescence in situ hybridization or RT-
PCR and immunophenotyping is required in order to assign all cases in to the right category. The aim of these techniques besides diagnosis is mainly to determine the prognosis of the leukemia. A major disadvantage of these methods, however, is that viable cells are necessary as the cells for genetic analysis have to divide in vitro in order to obtain metaphases for the analysis. Another problem is the long time of 72 hours from receipt of the material in the laboratory to obtain the result. Furthermore, great experience in preparation of chromosomes and even more in analyzing the karyotypes is required to obtain the correct result in at least 90% of cases. Using these techniques in combination, hematological malignancies in a first approach are separated into chronic myeloid leukemia (CML), chronic lymphatic (CLL), acute lymphoblastic (ALL), and acute myeloid leukemia (AML). Within the latter three disease entities several prognostically relevant subtypes have been established. As a second approach this further sub-classification is based mainly on genetic abnormalities of the leukemic blasts and clearly is associated with different prognoses.
The sub-classification of leukemias becomes increasingly important to guide therapy. The development of new, specific drugs and treatment approaches requires the identification of specific subtypes that may benefit from a distinct therapeutic protocol and, thus, can improve outcome of distinct subsets of leukemia. For example, the new therapeutic drug (STI571, Imatinib) inhibits the CML specific chimeric tyrosine kinase BCR-ABL generated from the genetic defect observed in
CML, the BCR-ABL-rearrangement due to the translocation between chromosomes 9 and 22 (t(9;22) (q34; qll)). In patients treated with this new drug, the therapy response is dramatically higher as compared to all other drugs that had been used so far. Another example is the subtype of acute myeloid leukemia AML M3 and its variant M3v both with karyotype t(15;17)(q22; qll-12). The introduction of a new drug (all-trans retinoic acid - ATRA) has improved the outcome in this subgroup of patient from about 50% to 85 % long-term survivors. As it is mandatory for these patients suffering from these specific leukemia subtypes to be identified as fast as possible so that the best therapy can be applied, diagnostics today must accomplish sub-classification with maximal precision. Not only for these subtypes but also for several other leukemia subtypes different treatment approaches could improve outcome. Therefore, rapid and precise identification of distinct leukemia subtypes is the future goal for diagnostics. Thus, the technical problem underlying the present invention was to provide means for leukemia diagnostics which overcome at least some of the disadvantages of the prior art diagnostic methods, in particular encompassing the time-consuming and unreliable combination of different methods and which provides a rapid assay to unambiguously distinguish one AML subtype from another, e.g. by genetic analysis.
According to Golub et al. (Science, 1999, 286, 531-7), gene expression profiles can be used for class prediction and discriminating AML from ALL samples. However, for the analysis of acute leukemias the selection of the two different subgroups was performed using exclusively morphologic-phenotypical criteria. This was only descriptive and does not provide deeper insights into the pathogenesis or the underlying biology of the leukemia. The approach reproduces only very basic knowledge of cytomorphology and intends to differentiate classes. The data is not sufficient to predict prognostically relevant cytogenetic aberrations.
Furthermore, the international application WO-A 03/039443 discloses marker genes the expression levels of which are characteristic for certain leukemia, e.g. AML subtypes and additionally discloses methods for differentiating between the subtype of AML cells by determining the expression profile of the disclosed marker genes. However, WO-A 03/039443 does not provide guidance which set of distinct genes discriminate between two subtypes and, as such, can be routineously taken in order to distinguish one AML subtype from another.
The problem is solved by the present invention, which provides a method for distinguishing MLL-PTD-positive AML from other AML subtypes in a sample, the method comprising determining the expression level of markers selected from the markers identifiable by their Affymetrix Identification Numbers (affy id) as defined in Tables 1, 2, and/or 3, wherein a lower expression of at least one polynucleotide defined by any of the numbers 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, and/or 50 of Table 1 is indicative for the presence of PTD (MLL-PTD-positive AML with normal karyotype) when PTD is distinguished from AML_NK (MLL- PTD-negative AML with normal karyotype), and/or wherein a lower expression of at least one polynucleotide defined by any of the numbers 1, 2, 3, 4, 5, 6, 7, 8, 9, 11, 12, 14, 15, 16, 18, 19, 20, 21, 22, 23, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 42, 44, 45, 47, 48, 49, and/or 50 of Table 2.1, and/or a higher expression of at least one polynucleotide defined by any of the numbers 10, 13, 17, 24, 25, 41, 43, and/or 46, of Table 2.1, is indicative for M4eo when M4eo is distinguished from all other subtypes, and/or wherein a lower expression of at least one polynucleotide defined by any of the numbers 1, 2, 3, 4, 6, 7, 8, 9, 10, 11, 12, 14, 15, 16, 17, 19, 20, 21, 22, 23, 24, 25, 26, 28, 29, 31, 32, 33, 34, 35, 36, 38, 39, 41, 42, 44, 45, 46, 48, 49, and/or 50 of Table 2.2, and/or a higher expression of 5, 13, 18, 27, 30, 37, 40, 43, and/or 47, of Table 2.2 is indicative for PTD when PTD is distinguished from all other subtypes, and/or wherein a lower expression of at least one polynucleotide defined by any of the numbers 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 49, and/or 50 of Table 2.3, and/or a higher expression of at least one polynucleotide defined by any of the numbers 34, and/or 48, of Table 2.3 is indicative for inv3 when inv3 is distinguished from all other subtypes, and/or wherein a lower expression of at least one polynucleotide defined by any of the numbers 1, 2, 3, 5, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 23, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 41, 42, 43, 44, 45, 46, 47, 48, and/or 50 of Table 2.4, and/or a higher expression of at least one polynucleotide defined by any of the numbers 4, 6, 7, 8, 22, 24, 40, and/or 49, of Table 2.4 is indicative for t(15;17) when t(15;17) is distinguished from all other subtypes, and/or wherein a lower expression of at least one polynucleotide defined by any of the numbers 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, and/or 50 of Table 2.5 is indicative for t(8;21) when t(8;21) is distinguished from all other subtypes, and/or wherein a lower expression of at least one polynucleotide defined by any of the numbers 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 13, 14, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 42, 43, 45, 46, 47, 48, 49, and/or 50 of Table 2.6, and/or a higher expression of at least one polynucleotide defined by any of the numbers 12, 15, 29, 41, and/or 44, of Table 2.6 is indicative for tMLL when tMLL is distinguished from all other subtypes, and/or wherein a lower expression of at least one polynucleotide defined by any of the numbers 1, 2, 4, 5, 7, 10, 12, 13, 16, 17, 19, 23, 25, 30, 31, 32, 33, 34, 37, 41, 43, 45, 47, 48, and/or 50 of Table 3.1,and/or a higher expression a polynucleotide defined by any of the numbers 3, 6, 8, 9, 11, 14, 15, 18, 20, 21, 22, 24, 26, 27, 28, 29, 35, 36, 38, 39, 40, 42, 44, 46, and/or 49, of Table 3.1, is indicative for M4eo when M4eo is distinguished from PTD, and/or wherein a lower expression of at least one polynucleotide defined by any of the numbers 5, 6, 9, 12, 23, 28, 38, 41, 44, 45, 46, and/or 47, of Table 3.2, and/or a higher expression of at least one polynucleotide defined by any of the numbers 1, 2, 3, 4, 7, 8, 10, 11, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 24, 25, 26, 27, 29, 30, 31, 32, 33, 34, 35, 36, 37, 39, 40, 42, 43, 48, 49, and/or 50 of Table 3.2, is indicative for M4eo when M4eo is distinguished from inv3, a lower expression of at least one polynucleotide defined by any of the numbers 2, 3, 4, 6, 11, 14, 20, 22, 26, 31, 32, 33, 34, 39, 40, 41, and/or 48, of Table 3.3, and/or a higher expression of at least one polynucleotide defined by any of the numbers 1, 5, 7, 8, 9, 10, 12, 13, 15, 16, 17, 18, 19, 21, 23, 24, 25, 27, 28, 29, 30, 35, 36, 37, 38, 42, 43, 44, 45, 46, 47, 49, and/or 50 of Table 3.3, is indicative for M4eo when M4eo is distinguished from t(15;17), and/or wherein a lower expression of at least one polynucleotide defined by any of the numbers 7, 31 , 40, and/or 49, of Table 3.4, and/or a higher expression of at least one polynucleotide defined by any of the numbers 1, 2, 3, 4, 5, 6, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 32, 33, 34, 35, 36, 37, 38, 39, 41, 42, 43, 44, 45, 46, 47, 48, and/or 50 of Table 3.4 is indicative for M4eo when M4eo is distinguished from t(8;21), and/or wherein a lower expression of at least one polynucleotide defined by any of the numbers 1, 3, 10, 14, 17, 18, 19, 21, 24, 25, 26, 31, 32, 34, 41, 44, and/or 50 of Table 3.5, and/or a higher expression of at least one polynucleotide defined by any of the numbers 2, 4, 5, 6, 7, 8, 9, 11, 12, 13, 15, 16, 20, 22, 23, 27, 28, 29, 30, 33, 35, 36, 37, 38, 39, 40, 42, 43, 45, 46, 47, 48, and/or 49, of Table 3.5 is indicative for M4eo when M4eo is distinguished from tMLL, and/or wherein a lower expression of at least one polynucleotide defined by any of the numbers 4, 6, 9, 28, 30, 32, 35, 37, 44, 45, and/or 48, of Table 3.6, and/or a higher expression of at least one polynucleotide defined by any of the numbers 1, 2, 3, 5, 7, 8, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 29, 31, 33, 34, 36, 38, 39, 40, 41, 42, 43, 46, 47, 49, and/or 50 of Table 3.6 is indicative for PTD when PTD is distinguished from inv3, and/or wherein a lower expression of at least one polynucleotide defined by any of the numbers 1, 2, 3, 4, 6, 7, 10, 11, 12, 13, 14, 15, 16, 17, 18, 20, 23, 27, 28, 29, 30, 31, 32, 33, 34, 36, 38, 39, 41, 43, 44, 45, 47, 48, and/or 50 of Table 3.7, and/or a higher expression of polynucleotide defined by any of the numbers 5, 8, 9, 19, 21, 22, 24, 25, 26, 35, 37, 40, 42, 46, and/or 49, of Table 3.7, is for PTD when PTD is distinguished from t(15;17), and/or wherein a lower expression of at least one polynucleotide defined by any of the numbers 7, 9, 10, 11, 13, 16, 20, 21, 22, 23, 30, 35, 36, 38, 42, 45, and/or 50 of Table 3.8, and/or a higher expression of at least one polynucleotide defined by any of the numbers 1, 2, 3, 4, 5, 6, 8, 12, 14, 15, 17, 18, 19, 24, 25, 26, 27, 28, 29, 31, 32, 33, 34, 37, 39, 40, 41, 43, 44, 46, 47, 48, and/or 49, of Table 3.8 is indicative for PTD when PTD is distinguished from t(8;21), and/or wherein a lower expression of at least one polynucleotide defined by any of the numbers 1, 5, 8, 10, 11, 13, 15, 17, 19, 25, 26, 28, 29, 34, and/or 46, of Table 3.9, and/or a higher expression of at least one polynucleotide defined by any of the numbers 2, 3, 4, 6, 7, 9, 12, 14, 16, 18, 20, 21, 22, 23, 24, 27, 30, 31, 32, 33, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 47, 48, 49, and/or 50 of Table 3.9 is indicative for PTD when PTD is distinguished from tMLL, and/or wherein a lower expression of at least one polynucleotide defined by any of the numbers 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 23, 24, 25, 26, 28, 29, 32, 33, 36, 38, 39, 40, 43, 44, 45, 46, 47, and/or 49, of Table 3.10, and/or a higher expression of at least one polynucleotide defined by any of the numbers 22, 27, 30, 31, 34, 35, 37, 41, 42, 48, and/or 50 of Table 3.10, is indicative for inv(3) when inv(3) is distinguished from t(15;17), and/or wherein a lower expression of at least one polynucleotide defined by any of the numbers 1, 5, 6, 9, 11, 12, 15, 17, 18, 19, 23, 27, 35, 36, 37, 39, 42, 43, 47, 49, and/or 50 of Table 3.11, and/or a higher expression of at least one polynucleotide defined by any of the numbers 2, 3, 4, 7, 8, 10, 13, 14, 16, 20, 21, 22, 24, 25, 26, 28, 29, 30, 31, 32, 33, 34, 38, 40, 41, 44, 45, 46, and/or 48, of Table 3.11 is indicative for inv(3) when inv(3) is distinguished from t(8;21), and/or wherein a lower expression of at least one polynucleotide defined by any of the numbers 1, 3, 4, 6, 7, 8, 12, 14, 15, 16, 17, 18, 19, 20, 21, 23, 25, 26, 28, 29, 30, 31, 33, 34, 35, 37, 38, 39, 42, 43, 44, 45, 47, 48, and/or 50 of Table 3.12, and/or a higher expression of at least one polynucleotide defined by any of the numbers 2, 5, 9, 10, 11, 13, 22, 24, 27, 32, 36, 40, 41, 46, and/or 49, of Table 3.12 is indicative for inv(3) when inv(3) is distinguished from tMLL, and/or wherein a lower expression of at least one polynucleotide defined by any of the numbers 3, 4, 7, 14, 16, 20, 22, 23, 24, 25, 26, 30, 35, 36, 37, 39, 40, 43, 44, 46, and or 50 of Table 3.13, and/or a higher expression of at least one polynucleotide defined by any of the numbers 1, 2, 5, 6, 8, 9, 10, 11, 12, 13, 15, 17, 18, 19, 21, 27, 28, 29, 31, 32, 33, 34, 38, 41, 42, 45, 47, 48, and/or 49 of Table 3.13, is indicative for t(15;17) when t(15;17) is distinguished from t(8;21), and/or wherein a lower expression of at least one polynucleotide defined by any of the numbers 13, 15, 25, 26, 27, 28, 30, 32, 33, 35, 36, 38, 39, 43, 48, and/or 49, of Table 3.14, and/or a higher expression of at least one polynucleotide defined by any of the numbers 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 14, 16, 17, 18, 19, 20, 21, 22, 23, 24, 29, 31, 34, 37, 40, 41, 42, 44, 45, 46, 47, and/or 50 of Table 3.14, is indicative for t(15;17) when t(15;17) is distinguished from tMLL, and/or wherein a lower expression of at least one polynucleotide defined by any of the numbers 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 13, 15, 16, 18, 19, 21, 23, 24, 25, 26, 27, 28, 29, 30, 32, 33, 34, 35, 36, 38, 39, 40, 41, 42, 43, 44, 47, 48, of Table 3.15, and/or a higher expression of at least one polynucleotide defined by any of the numbers 12, 14, 17, 20, 22, 31, 37, 45, 46, 49, and/or 50 of Table 3.15, is indicative for t(8;21) when t(8;21) is distinguished from tMLL.
As used herein, the following definitions apply to the above used abbreviations (see also example 1): tMLL: AML with translocations in the MLL gene (t(l Iq23)/MLL)
PTD: AML with normal karyotype and Partial Tandem Duplication (PTD) within the MLL gene (MLL-PTD) AMLJSfK AML with normal karyotype (no Partial Tandem Duplication (PTD) within the MLL gene) t(8;21) AML with translocation t(8;21) t(15;17) AML with translocation t(15;17) t(inv3) AML with inversion 3
M4eo AML with inversion 16 (inv( 16))
As used herein, "all other subtypes" refer to the subtypes of the present invention, i.e. if one subtype is distinguished from "all other subtypes", it is distiguished from all other subtypes contained in the present invention.
According to the present invention, a "sample" means any biological material containing genetic information in the form of nucleic acids or proteins obtainable or obtained from an individual. The sample includes e.g. tissue samples, cell samples, bone marrow and/or body fluids such as blood, saliva, semen. Preferably, the sample is blood or bone marrow, more preferably the sample is bone marrow. The person skilled in the art is aware of methods, how to isolate nucleic acids and proteins from a sample. A general method for isolating and preparing nucleic acids from a sample is outlined in Example 3.
According to the present invention, the term "lower expression" is generally assigned to all by numbers and Affymetrix Id. definable polynucleotides the t- values and fold change (fc) values of which are negative, as indicated in the Tables. Accordingly, the term "higher expression" is generally assigned to all by numbers and Affymetrix Id. definable polynucleotides the t-values and fold change (fc) values of which are positive.
According to the present invention, the term "expression" refers to the process by which mRNA or a polypeptide is produced based on the nucleic acid sequence of a gene, i.e. „expression" also includes the formation of mRNA upon transcription. In accordance with the present invention, the term determining the expression level" preferably refers to the determination of the level of expression, namely of the markers.
Generally, "marker" refers to any genetically controlled difference which can be used in the genetic analysis of a test versus a control sample, for the purpose of assigning the sample to a defined genotype or phenotype. As used herein, "markers" refer to genes which are differentially expressed in, e.g., different AML subtypes. The markers can be defined by their gene symbol name, their encoded protein name, their transcript identification number (cluster identification number), the data base accession number, public accession number or GenBank identifier or, as done in the present invention, Affymetrix identification number, chromosomal location, UniGene accession number and cluster type, LocusLink accession number (see Examples and Tables).
The Affymetrix identification number (affy id) is accessible for anyone and the person skilled in the art by entering the "gene expression omnibus" internet page of the National Center for Biotechnology Information (NCBI) (http://www.ncbi.nlm.nih.gov/geo/). In particular, the affy id's of the polynucleotides used for the method of the present invention are derived from the so-called U133 chip. The sequence data of each identification number can be viewed at http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GPL96
Generally, the expression level of a marker is determined by the determining the expression of its corresponding "polynucleotide" as described hereinafter.
According to the present invention, the term „polynucleotide" refers, generally, to a DNA, in particular cDNA, or RNA, in particular a cRNA, or a portion thereof or a polypeptide or a portion thereof. In the case of RNA (or cDNA), the polynucleotide is formed upon transcription of a nucleotide sequence which is capable of expression. The polynucleotide fragments refer to fragments preferably of between at least 8, such as 10, 12, 15 or 18 nucleotides and at least 50, such as 60, 80, 100, 200 or 300 nucleotides in length, or a complementary sequence thereto, representing a consecutive stretch of nucleotides of a gene, cDNA or mRNA. In other terms, polynucleotides include also any fragment (or complementary sequence thereto) of a sequence derived from any of the markers defined above as long as these fragments unambiguously identify the marker.
The determination of the expression level may be effected at the transcriptional or translational level, i.e. at the level of mRNA or at the protein level. Protein fragments such as peptides or polypeptides advantageously comprise between at least 6 and at least 25, such as 30, 40, 80, 100 or 200 consecutive amino acids representative of the corresponding full length protein. Six amino acids are generally recognized as the lowest peptidic stretch giving rise to a linear epitope recognized by an antibody, fragment or derivative thereof. Alternatively, the proteins or fragments thereof may be analysed using nucleic acid molecules specifically binding to three-dimensional structures (aptamers).
Depending on the nature of the polynucleotide or polypeptide, the determination of the expression levels may be effected by a variety of methods. For determining and detecting the expression level, it is preferred in the present invention that the polynucleotide, in particular the cRNA, is labelled.
The labelling of the polynucleotide or a polypeptide can occur by a variety of methods known to the skilled artisan. The label can be fluorescent, chemiluminescent, bioluminescent, radioactive (such as 3H or 32P). The labelling compound can be any labelling compound being suitable for the labelling of polynucleotides and/or polypeptides. Examples include fluorescent dyes, such as fluorescein, dichlorofluorescein, hexachlorofluorescein, BODIPY variants, ROX, tetramethylrhodamin, rhodamin X, Cyanine-2, Cyanine-3, Cyanine-5, Cyanine-7,
IRD40, FluorX, Oregon Green, Alexa variants (available e.g. from Molecular Probes or Amersham Biosciences) and the like, biotin or biotinylated nucleotides, digoxigenin, radioisotopes, antibodies, enzymes and receptors. Depending on the type of labelling, the detection is done via fluorescence measurements, conjugation to streptavidin and/or avidin, antigen-antibody- and/or antibody- ntibody- interactions, radioactivity measurements, as well as catalytic and or receptor/ligand interactions. Suitable methods include the direct labelling (incorporation) method, the amino-modified (amino-allyl) nucleotide method (available e.g. from Ambion), and the primer tagging method (DNA dendrimer labelling, as kit available e.g. from Genisphere). Particularly preferred for the present invention is the use of biotin or biotinylated nucleotides for labelling, with the latter being directly incorporated into, e.g. the cRNA polynucleotide by in vitro transcription.
If the polynucleotide is mRNA, cDNA may be prepared into which a detectable label, as exemplified above, is incorporated. Said detectably labelled cDNA, in single-stranded form, may then be hybridised, preferably under stringent or highly stringent conditions to a panel of single-stranded oligonucleotides representing different genes and affixed to a solid support such as a chip. Upon applying appropriate washing steps, those cDNAs will be detected or quantitatively detected that have a counterpart in the oligonucleotide panel. Various advantageous embodiments of this general method are feasible. For example, the mRNA or the cDNA may be amplified e.g. by polymerase chain reaction, wherein it is preferable, for quantitative assessments, that the number of amplified copies conesponds relative to further amplified mRNAs or cDNAs to the number of mRNAs originally present in the cell. In a preferred embodiment of the present in ivention, the cDNAs are transcribed into cRNAs prior to the hybridisation step wherein only in the transcription step a label is incorporated into the nucleic acid and wherein the cRNA is employed for hybridisation. Alternatively, the label may be attached subsequent to the transcription step.
Similarly, proteins from a cell or tissue under investigation may be contacted with a panel of aptamers or of antibodies or fragments or derivatives thereof. The antibodies etc. may be affixed to a solid support such as a chip. Binding of proteins indicative of an AML subtype may be verified by binding to a detectably labelled secondary antibody or aptamer. For the labelling of antibodies, it is referred to
Harlow and Lane, "Antibodies, a laboratory manual", CSH Press, 1988, Cold Spring Harbor. Specifically, a minimum set of proteins necessary for diagnosis of all AML subtypes may be selected for creation of a protein array system to make diagnosis on a protein lysate of a diagnostic bone marrow sample directly. Protein Array Systems for the detection of specific protein expression profiles already are available (for example: Bio-Plex, BIORAD, Munchen, Germany). For this application preferably antibodies against the proteins have to be produced and immobilized on a platform e.g. glasslides or microtiterplates. The immobilized antibodies can be labelled with a reactant specific for the certain target proteins as discussed above. The reactants can include enzyme substrates, DNA, receptors, antigens or antibodies to create for example a capture sandwich irnmunoassay.
For reliably distinguishing MLL-PTD-positive AML from other AML subtypes in a sample it is useful that the expression of more than one of the above defined markers is determined. As a criterion for the choice of markers, the statistical significance of markers as expressed in q or p values based on the concept of the false discovery rate is determined. In doing so, a measure of statistical significance called the q value is associated with each tested feature. The q value is similar to the p value, except it is a measure of significance in terms of the false discovery rate rather than the false positive rate (Storey JD and Tibsbirani R. Proc.Natl.Acad.Sci., 2003, Vol. 100:9440-5.
In a preferred embodiment of the present invention, markers as defined in Table 1.1-3.15 having a q- value of less than 3E-03, more preferred less than 1.5E-09, most preferred less than 1.5E-11, less than 1.5E-20, less than 1.5E-30, are measured.
Of the above defined markers, the expression level of at least two, preferably of at least ten, more preferably of at least 25, most preferably of 5O of at least one of the
Tables of the markers is determined.
In another preferred embodiment, the expression level of at least 2, of at least 5, of at least 10 out of the markers having the numbers 1 - 10, 1-20, 1-40, 1-50 of at least one of the Tables are measured.
The level of the expression of the „marker", i.e. the expression of the polynucleotide is indicative of the AML subtype of a cell or an organism. The level of expression of a marker or group of markers is measured and is compared with the level of expression of the same marker or the same group of markers from other cells or samples. The comparison may be effected in an actual experiment or in silico. When the expression level also referred to as expression pattern or expression signature (expression profile) is measurably different, there is according to the invention a meaningful difference in the level of expression. Preferably the difference at least is 5 %, 10% or 20%, more preferred at least 50% or may even be as high as 75% or 100%. More prefened the difference in the level of expression is at least 200%, i.e. two fold, at least 500%, i.e. five fold, or at least 1000%, i.e. 10 fold.
Accordingly, the expression level of markers expressed lower in a first subtype than in at least one second subtype, which differs from the first subtype, is at least
5 %, 10% or 20%, more preferred at least 50% or may even be 75% or 100%, i.e. 2-fold lower, preferably at least 10-fold, more preferably at least 50-fold, and most preferably at least 100-fold lower in the first subtype. On the other hand, the expression level of markers expressed higher in a first subtype than in at least one second subtype, which differs from the first subtype, is at least 5 %, 10% or 20%, more preferred at least 50% or may even be 75% or 100%, i.e. 2-fold higher, preferably at least 10-fold, more preferably at least 50-fold, and most preferably at least 100-fold higher in the first subtype.
In another embodiment of the present invention, the sample is derived from an individual having leukaemia, preferably AML.
For the method of the present invention it is prefened if the polynucleotide the expression level of which is determined is in form of a transcribed polynucleotide. A particularly preferred transcribed polynucleotide is an mRNA, a cDNA and/or a cRNA, with the latter being preferred. Transcribed polynucleotides are isolated from a sample, reverse transcribed and/or amplified, and labelled, by employing methods well-known the person skilled in the art (see Example 3). In a preferred embodiment of the methods according to the invention, the step of determining the expression profile further comprises amplifying the transcribed polynucleotide.
In order to determine the expression level of the transcribed polynucleotide by the method of the present invention, it is preferred that the method comprises hybridizing the transcribed polynucleotide to a complementary polynucleotide, or a portion thereof, under stringent hybridization conditions, as described hereinafter.
The term "hybridizing" means hybridization under conventional hybridization conditions, preferably under stringent conditions as described, for example, in Sambrook, J., et al., in "Molecular Cloning: A Laboratory Manual" (1989), Eds. J. Sambrook, E. F. Fritsch and T. Maniatis, Cold Spring Harbour Laboratory Press,
Cold Spring Harbour, NY and the further definitions provided above. Such conditions are, for example, hybridization in 6x SSC, pH 7.0 / 0.1% SDS at about 45°C for 18-23 hours, followed by a washing step with 2x SSC/0.1% SDS at 50°C. In order to select the stringency, the salt concentration in the washing step can for example be chosen between 2x SSC/0.1% SDS at room temperature for low stringency and 0.2x SSC/0.1% SDS at 50°C for high stringency. In addition, the temperature of the washing step can be varied between room temperature, ca. 22°C, for low stringency, and 65°C to 70° C for high stringency. Also contemplated are polynucleotides that hybridize at lower stringency hybridization conditions. Changes in the stringency of hybridization and signal detection are primarily accomplished through the manipulation, preferably of formamide concentration
(lower percentages of formamide result in lowered stringency), salt conditions, or temperature. For example, lower stringency conditions include an overnight incubation at 37°C in a solution comprising 6X SSPE (20X SSPE = 3M NaCl; 0.2M NaH2PO4; 0.02M EDTA, pH 7.4), 0.5% SDS, 30% formamide, 100 mg/ml salmon sperm blocking DNA, followed by washes at 50°C with 1 X SSPE, 0.1 %
SDS. In addition, to achieve even lower stringency, washes performed following stringent hybridization can be done at higher salt concentrations (e.g. 5x SSC). Variations in the above conditions may be accomplished through the inclusion and/or substitution of alternate blocking reagents used to suppress background in hybridization experiments. The inclusion of specific blocking reagents may require modification of the hybridization conditions described above, due to problems with compatibility.
"Complementary" and "complementarity", respectively, can be described by the percentage, i.e. proportion, of nucleotides which can form base pairs between two polynucleotide strands or within a specific region or domain of the two strands.
Generally, complementary nucleotides are, according to the base pairing rules, adenine and thymine (or adenine and uracil), and cytosine and guanine.
Complementarity may be partial, in which only some of the nucleic acids' bases are matched according to the base pairing rules. Or, there may be a complete or total complementarity between the nucleic acids. The degree of complementarity between nucleic acid strands has effects on the efficiency and strength of hybridization between nucleic acid strands.
Two nucleic acid strands are considered to be 100% complementary to each other over a defined length if in a defined region all adenines of a first strand can pair with a thymine (or an uracil) of a second strand, all guanines of a first strand can pair with a cytosine of a second strand, all thymine (or uracils) of a first strand can pair with an adenine of a second strand, and all cytosines of a first strand can pair with a guanine of a second strand, and vice versa. According to the present invention, the degree of complementarity is determined over a stretch of 20, preferably 25, nucleotides, i.e. a 60% complementarity means that within a region of 20 nucleotides of two nucleic acid strands 12 nucleotides of the first strand can base pair with 12 nucleotides of the second strand according to the above ruling, either as a stretch of 12 contiguous nucleotides or interspersed by non-pairing nucleotides, when the two strands are attached to each other over said region of 20 nucleotides. The degree of complementarity can range from at least about 50% to full, i.e. 100% complementarity. Two single nucleic acid strands are said to be "substantially complementary" when they are at least about 80% complementary, preferably about 90% or higher. For carrying out the method of the present invention substantial complementarity is preferred.
Prefened methods for detection and quantification of the amount of polynucleotides, i.e. for the methods according to the invention allowing the determination of the level of expression of a marker, are those described by Sambrook et al. (1989) or real time methods known in the art as the TaqMan® method disclosed in WO92/02638 and the corresponding U.S. 5,210,015, U.S. 5,804,375, U.S. 5,487,972. This method exploits the exonuclease activity of a polymerase to generate a signal. In detail, the (at least one) target nucleic acid component is detected by a process comprising contacting the sample with an oligonucleotide containing a sequence complementary to a region of the target nucleic acid component and a labeled oligonucleotide containing a sequence complementary to a second region of the same target nucleic acid component sequence strand, but not including the nucleic acid sequence defined by the first oligonucleotide, to create a mixture of duplexes during hybridization conditions, wherein the duplexes comprise the target nucleic acid annealed to the first oligonucleotide and to the labeled oligonucleotide such that the 3 '-end of the first oligonucleotide is adjacent to the 5 '-end of the labeled oligonucleotide. Then this mixture is treated with a template-dependent nucleic acid polymerase having a 5' to 3' nuclease activity under conditions sufficient to permit the 5' to 3' nuclease activity of the polymerase to cleave the annealed, labeled oligonucleotide and release labeled fragments. The signal generated by the hydrolysis of the labeled oligonucleotide is detected and/ or measured. TaqMan® technology eliminates the need for a solid phase bound reaction complex to be formed and made detectable. Other methods include e.g. fluorescence resonance energy transfer between two adjacently hybridized probes as used in the LightCycler® format described in U.S. 6,174,670.
A prefened protocol if the marker, i.e. the polynucleotide, is in form of a transcribed nucleotide, is described in Example 3, where total RNA is isolated, cDNA and, subsequently, cRNA is synthesized and biotin is incorporated during the transcription reaction. The purified cRNA is applied to commercially available arcays which can be obtained e.g. from Affymetrix. The hybridized cRNA is detected according to the methods described in Example 3. The anays are produced by photolithography or other methods known to experts skilled in the art e.g. from U.S. 5,445,934, U.S. 5,744,305, U.S. 5,700,637, U.S. 5,945,334 and EP 0 619 321 or EP 0373 203, or as decribed hereinafter in greater detail.
In another embodiment of the present invention, the polynucleotide or at least one of the polynucleotides is in form of a polypeptide. In another prefened embodiment, the expression level of the polynucleotides or polypeptides is detected using a compound which specifically binds to the polynucleotide of the polypeptide of the present invention.
As used herein, "specifically binding" means that the compound is capable of discriminating between two or more polynucleotides or polypeptides, i.e. it binds to the desired polynucleotide or polypeptide, but essentially does not bind unspecifically to a different polynucleotide or polypeptide.
The compound can be an antibody, or a fragment thereof, an enzyme, a so-called small molecule compound, a protein-scaffold, preferably an anticalin. In a prefened embodiment, the compound specifically binding to the polynucleotide or polypeptide is an antibody, or a fragment thereof.
As used herein, an "antibody" comprises monoclonal antibodies as first described by Kohler and Milstein in Nature 278 (1975), 495-497 as well as polyclonal antibodies, i.e. entibodies contained in a polyclonal antiserum. Monoclonal antibodies include those produced by transgenic mice. Fragments of antibodies include F(ab')2, Fab and Fv fragments. Derivatives of antibodies include scFvs, chimeric and humanized antibodies. See, for example Harlow and Lane, loc. cit. For the detection of polypeptides using antibodies or fragments thereof, the person skilled in the art is aware of a variety of methods, all of which are included in the present invention. Examples include immunoprecipitation, Western blotting, Enzyme-linked immuno sorbent assay (ELISA), Enzyme-linked imrnuno sorbent assay (RIA), dissociation-enhanced lanthanide fluoro immuno assay (DELFIA), scintillation proximity assay (SPA). For detection, it is desirable if the antibody is labelled by one of the labelling compounds and methods described supra.
In another prefened embodiment of the present invention, the method for distinguishing MLL-PTD-positive AML from other AML subtypes is canied out on an anay.
In general, an "anay" or "microanay" refers to a linear or two- or three dimensional anangement of preferably discrete nucleic acid or polypeptide probes which comprises an intentionally created collection of nucleic acid or polypeptide probes of any length spotted onto a substrate/solid support. The person skilled in the art knows a collection of nucleic acids or polypeptide spotted onto a substrate/solid support also under the term "anay". As known to the person skilled in the art, a microanay usually refers to a miniaturised anay anangement, with the probes being attached to a density of at least about 10, 20, 50, 100 nucleic acid molecules refening to different or the same genes per cm . Furthermore, where appropriate an anay can be refened to as "gene chip". The anay itself can have different formats, e.g. libraries of soluble probes or libraries of probes tethered to resin beads, silica chips, or other solid supports.
The process of anay fabrication is well-known to the person skilled in the art. In the following, the process for preparing a nucleic acid anay is described. Commonly, the process comprises preparing a glass (or other) slide (e.g. chemical treatment of the glass to enhance binding of the nucleic acid probes to the glass surface), obtaining DNA sequences representing genes of a genome of interest, and spotting sequences these sequences of interest onto glass slide. Sequences of interest can be obtained via creating a cDNA library from an mRNA source or by using publicly available databases, such as GeneBank, to annotate the sequence info mation of custom cDNA libraries or to identify cDNA clones from previously prepared libraries. Generally, it is recommendable to amplify obtained sequences by PCR in order to have sufficient amounts of DNA to print on the anay. The liquid containing the amplified probes can be deposited on the anay by using a set of microspotting pins. Ideally, the amount deposited should be uniform. The process can further include UV-crosslinking in order to enhance immobilization of the probes on the anay.
In a prefened embodiment, the anay is a high density oligonucleotide (oligo) anay using a light-directed chemical synthesis process, employing the so-called photolithography technology. Unlike common cDNA anays, oligo anays (according to the Affymetrix technology) use a single-dye technology. Given the sequence information of the markers, the sequence can be synthesized directly onto the anay, thus, bypassing the need for physical intermediates, such as PCR products, required for making cDNA anays. For this purpose, the marker, or partial sequences thereof, can be represented by 14 to 20 features, preferably by less than 14 features, more preferably less than 10 features, even more preferably by 6 features or less, with each feature being a short sequence of nucleotides (oligonucleotide), which is a perfect match (PM) to a segment of the respective gene. The PM oligonucleotide are paired with mismatch (MM) oligonucleotides which have a single mismatch at the central base of the nucleotide and are used as "controls". The chip exposure sites are defined by masks and are deprotected by the use of light, followed by a chemical coupling step resulting in the synthesis of one nucleotide. The masking, light deprotection, and coupling process can then be repeated to synthesize the next nucleotide, until the nucleotide chain is of the specified length.
Advantageously, the method of the present invention is canied out in a robotics system including robotic plating and a robotic liquid transfer system, e.g. using microfluidics, i.e. channelled structured.
A particular prefened method according to the present invention is as follows:
1. Obtaining a sample, e.g. bone manow aliquots, from a patient having AML
2. Extracting RNA, preferably mRNA, from the sample 3. Reverse transcribing the RNA into cDNA
4. In vitro transcribing the cDNA into cRNA
5. Fragmenting the cRNA 6. Hybridizing the fragmented cRNA on standard microanays
7. Determining hybridization
In another embodiment, the present invention is directed to the use of at least one marker selected from the markers identifiable by their Affymetrix Identification
Numbers (affy id) as defined in Tables 1, 2, and/or 3, for the manufacturing of a diagnostic for distinguishing MLL-PTD-positive AML from other AML subtypes. The use of the present invention is particularly advantageous for distinguishing MLL-PTD-positive AML from other AML subtypes in an individual having AML. The use of said markers for diagnosis of MLL-PTD-positive AML, preferably based on microanay technology, offers the following advantages: (1) more rapid and more precise diagnosis, (2) easy to use in laboratories without specialized experience, (3) abolishes the requirement for analyzing viable cells for chromosome analysis (transport problem), and (4) very experienced hematologists for cytomorphology and cytochemistry, immunophenotyping as well as cytogeneticists and molecularbiologists are no longer required.
Accordingly, the present invention refers to a diagnostic kit containing at least one marker selected from the markers identifiable by their Affymetrix Identification Numbers (affy id) as defined in Tables 1, and/or 3 for distinguishing MLL-PTD- positive AML from other AML subtypes, in combination with suitable auxiliaries. Suitable auxiliaries, as used herein, include buffers, enzymes, labelling compounds, and the like. In a prefened embodiment, the marker contained in the kit is a nucleic acid molecule which is capable of hybridizing to the mRNA conesponding to at least one marker of the present invention. Preferably, the at least one nucleic acid molecule is attached to a solid support, e.g. a polystyrene microtiter dish, nitrocellulose membrane, glass surface or to non-immobilized particles in solution.
In another prefened embodiment, the diagnostic kit contains at least one reference for a MLL-PTD-positive AML subtype. As used herein, the reference can be a sample or a data bank.
In another embodiment, the present invention is directed to an apparatus for distinguishing MLL-PTD-positive AML from other AML subtypes in a sample, containing a reference data bank obtainable by comprising (a) compiling a gene expression profile of a patient sample by determining the expression level at least one marker selected from the markers identifiable by their Affymetrix Identification Numbers (affy id) as defined in Tables 1, and/or 3, and (b) classifying the gene expression profile by means of a machine learning algorithm.
According to the present invention, the "machine learning algorithm" is a computational-based prediction methodology, also known to the person skilled in the art as "classifier", employed for characterizing a gene expression profile. The signals conesponding to a certain expression level which are obtained by the microanay hybridization are subjected to the algorithm in order to classify the expression profile. Supervised learning involves "training" a classifier to recognize the distinctions among classes and then "testing" the accuracy of the classifier on an independent test set. For new, unknown sample the classifier shall predict into which class the sample belongs.
Preferably, the machine learning algorithm is selected from the group consisting of Weighted Voting, K-Nearest Neighbors, Decision Tree Induction, Support Vector Machines (SVM), and Feed-Forward Neural Networks. Most preferably, the machine learning algorithm is Support Vector Machine, such as polynomial kernel and Gaussian Radial Basis Function-kernel SVM models.
The classification accuracy of a given gene list for a set of microanay experiments is preferably estimated using Support Vector Machines (SVM), because there is evidence that SVM-based prediction slightly outperforms other classification techniques like k-Nearest Neighbors (k-NN). The LIBSVM software package version 2.36 was used (SVM-type: C-SVC, linear kernel (http://www.csie.ntu.edu.tw/~cjlin/libsvm/)). The skilled artisan is furthermore refened to Brown et al., Proc.Natl.Acad.Sci., 2000; 97: 262-267, Furey et al, Bioinformatics. 2000; 16: 906-914, and Vapnik V. Statistical Learning Theory. New York: Wiley, 1998.
In detail, the classification accuracy of a given gene list for a set of microanay experiments can be estimated using Support Vector Machines (SVM) as supervised learning technique. Generally, SVMs are trained using differentially expressed genes which were identified on a subset of the data and then this trained model is employed to assign new samples to those trained groups from a second and different data set. Differentially expressed genes were identified applying ANOVA and t-test-statistics (Welch t-test). Based on identified distinct gene expression signatures respective training sets consisting of 2/3 of cases and test sets with 1/3 of cases to assess classification accuracies are designated. Assignment of cases to training and test set is randomized and balanced by diagnosis. Based on the training set a Support Vector Machine (SVM) model is built.
According to the present invention, the apparent accuracy, i.e. the overall rate of conect predictions of the complete data set was estimated by lOfold cross validation. This means that the data set was divided into 10 approximately equally sized subsets, an SVM-model was trained for 9 subsets and predictions were generated for the remaining subset. This training and prediction process was repeated 10 times to include predictions for each subset. Subsequently the data set was split into a training set, consisting of two thirds of the samples, and a test set with the remaining one third. Apparent accuracy for the training set was estimated by lOfold cross validation (analogous to apparent accuracy for complete set). A SVM-model of the training set was built to predict diagnosis in the independent test set, thereby estimating true accuracy of the prediction model. This prediction approach was applied both for overall classification (multi-class) and binary classification (diagnosis X => yes or no). For the latter, sensitivity and specificity were calculated:
Sensitivity = (number of positive samples predicted)/(number of true positives)
Specificity = (number of negative samples predicted)/(number of true negatives)
In a prefened embodiment, the reference data bank is backed up on a computational data memory chip which can be inserted in as well as removed from the apparatus of the present invention, e.g. like an interchangeable module, in order to use another data memory chip containing a different reference data bank.
The apparatus of the present invention containing a desired reference data bank can be used in a way such that an unknown sample is, first, subjected to gene expression profiling, e.g. by microanay analysis in a manner as described supra or in the art, and the expression level data obtained by the analysis are, second, fed into the apparatus and compared with the data of the reference data bank obtainable by the above method. For this purpose, the apparatus suitably contains a device for entering the expression level of the data, for example a control panel such as a keyboard. The results, whether and how the data of the unknown sample fit into the reference data bank can be made visible on a provided monitor or display screen and, if desired, printed out on an incorporated of connected printer.
Alternatively, the apparatus of the present invention is equipped with particular appliances suitable for detecting and measuring the expression profile data and, subsequently, proceeding with the comparison with the reference data bank. In this embodiment, the apparatus of the present invention can contain a gripper arm and/or a tray which takes up the microanay containing the hybridized nucleic acids.
In another embodiment, the present invention refers to a reference data bank for distinguishing MLL-PTD-positive AML from other AML subtypes in a sample obtainable by comprising (a) compiling a gene expression profile of a patient sample by determining the expression level of at least one marker selected from the markers identifiable by their Affymetrix Identification Numbers (affy id) as defined in Tables 1, and/or 3, and (b) classifying the gene expression profile by means of a machine learning algorithm.
Preferably, the reference data bank is backed up and/or contained in a computational memory data chip.
The invention is further illustrated in the following table and examples, without limiting the scope of the invention:
TABLE 1.1-3.15
Table 1.1-3.15 show AML subtype analysis of MLL-PTD-positive AML versus other AML subtypes. The analysed markers are ordered according to their q-values, beginning with the lowest q-values.
For convenience and a better understanding, Tables 1.1 to 3.15 are accompanied with explanatory tables (Table 1.1 A to 3.15 A) where the numbering and the
Affymetrix Id are further defined by other parameters, e.g. gene bank accession number. EXAMPLES
Example 1: General experimental design of the invention and results
Partial tandem duplication within the MLL-gene (MLL-PTD) can be found in 10% of AML with normal karyotype. Like MLL-translocations (t(llq23)/MLL) the occurence of MLL-PTD is characterized by an unfavourable prognosis. The pathogenetic mechanisms of the MLL-PTD are poorly understood and downstream genes effected by this molecular abenation are not known. To get more insight into the pathogenesis of PTD+ AML we performed global gene expression profiling of 184 AML samples at diagnosis using the U133 set of expression microanays (Affymetrix) with >30,000 human genes represented on both anays. Microanay data was analyzed by pattern recognition algorithms (Principal Component Analysis (PCA), hierarchical clustering), as well as Support Vector Machines
(SVM) for estimation of classification accuracies. Therefore, all samples were divided into a training set consisting of 2/3 of cases to built a SVM model and a test set with remaining 1/3 of cases. Assignment of cases to training and test set was randomized and balanced by diagnosis. Differentially expressed genes were selected according to ANOVA and t-test-statistics in the training set. Classification accuracy was assessed in the test set. In detail, we analyzed 30 cases with t(l lq23)/MLL, 30 cases with normal karyotype AML and MLL-PTD (PTD+ AML) and 124 cases with normal karyotype without MLL-PTD (AML-NK). All data analysis algorithms demonstrate that PTD+ AML can clearly be distinguished from t(l Iq23)/MLL positive AML with 100% accuracy. Thus, despite an identical gene targeted by molecular mutation or chromosomal translocation, this finding illustrates that both kinds of abenations lead to biologically distinct leukemia subclasses. Some of the most significantly differentially expressed genes that were highly expressed in t(l lq23)/MLL in comparison to PTD+ AML were CACNA2DA, MBNL1, and PBX3. Reversely, genes with high expression in
PTD+ and low in t(l lq23)/MLL samples were HOXB5, HOXB2, MAN1A1, and ZNF207. At next, we addressed the question whether PTD+ AML can be discriminated from AML-NK by a specific gene expression signature. Both PCA and hierarchical cluster visualize that the MLL-PTD samples characterize a homogeneous subgroup within AML with normal karyotype, but do not separate from them. Some of the genes that were highly expressed in AML-NK and low in PTD+ were AAK1, RAB4A, HOXA2, BID. On the other hand genes that were low in AML-NK and high in PTD+ were, among others, MLL, YY1, and SRP46. In addition, we attempted to classify the analyzed samples by means of SVM. Here, the training set comprised 83 AML-NK and 19 PTD+ AML cases, the test set 41 AML-NK and 9 PTD+ AML cases, respectively. The 50 test samples were assigned to the conect group with an accuracy of 88%. In detail, 6/9 PTD+ AML
(92.7% specificity, 66.7% sensitivity) and 38/41 AML-NK (66.7% specificity, 92.7% sensitivity) were accurately assigned. In conclusion, despite a significantly worse prognosis of the PTD+ AML cases within the large group of AML with normal karyotype it is not possible to designate a highly characteristic specific gene expression signature at diagnosis as has been demonstrated for AML with balanced chromosomal abenations. This unexpected results may be in part due to the fact that pts with PTD do not belong to a specific morphologic subgroup. Thus the expression pattern associated with heterogenous FAB subtypes may overwrite that generated bei the PTD. In addition, different unknown accompanying mutation may generate a dominant expression pattern.
Example 2: General materials, methods and definitions of functional annotations
The methods section contains both information on statistical analyses used for identification of differentially expressed genes and detailed annotation data of identified microanay probesets.
Affymetrix Probeset Annotation All annotation data of GeneChip® anays are extracted from the NetAffx™
Analysis Center (internet website: www.affymetrix.com). Files for U133 set anays, including U133A and U133B microanays are derived from the June 2003 release. The original publication refers to: Liu G, Loraine AE, Shigeta R, Cline M, Cheng J, Valmeekam V, Sun S, Kulp D, Siani-Rose MA. NetAffx: Affymetrix probesets and annotations. Nucleic Acids Res. 2003;3 l(l):82-6.
The sequence data are omitted due to their large size, and because they do not change, whereas the annotation data are updated periodically, for example new information on chromomal location and functional annotation of the respective gene products. Sequence data are available for download in the NetAffx Download
Center (www.affymetrix.com) Data fields:
In the following section, the content of each field of the data files are described. Microanay probesets, for example found to be differentially expressed between different types of leukemia samples are further described by additional information.
The fields are of the following types:
1. GeneChip Anay Information
2. Probe Design Information 3. Public Domain and Genomic References
1. GeneChip Anay information
HG-U133 ProbeSet D: HG-U133 ProbeSetJD describes the probe set identifier. Examples are:
200007_at, 20001 l_s_at, 200012_x_at.
GeneChip:
The description of the GeneChip probe anay name where the respective probeset is represented. Examples are: Affymetrix Human Genome U133A Anay or
Affymetrix Human Genome U133B Anay.
2. Probe Design Information
Sequence Type:
The Sequence Type indicates whether the sequence is an Exemplar, Consensus or Control sequence. An Exemplar is a single nucleotide sequence taken directly from a public database. This sequence could be an mRNA or EST. A Consensus sequence, is a nucleotide sequence assembled by Affymetrix, based on one or more sequence taken from a public database.
Transcript ID:
The cluster identification number with a sub-cluster identifier appended.
Sequence Derived From: The accession number of the single sequence, or representative sequence on which the probe set is based. Refer to the "Sequence Source" field to determine the database used.
Sequence ID:
For Exemplar sequences: Public accession number or GenBank identifier. For Consensus sequences: Affymetrix identification number or public accession number.
Sequence Source:
The database from which the sequence used to design this probe set was taken. Examples are: GenBank®, RefSeq, UniGene, TIGR (annotations from The Institute for Genomic Research).
3. Public Domain and Genomic References
Most of the data in this section come from LocusLink and UniGene databases, and are annotations of the reference sequence on which the probe set is modeled.
Gene Symbol and Title:
A gene symbol and a short title, when one is available. Such symbols are assigned by different organizations for different species. Affymetrix annotational data come from the UniGene record. There is no indication which species-specific databank was used, but some of the possibilities include for example HUGO: The Human Genome Organization.
MapLocation:
The map location describes the chromosomal location when one is available.
Unigene_Accession:
UniGene accession number and cluster type. Cluster type can be "full length" or "est", or " — " if unknown.
LocusLink: This information represents the LocusLink accession number. Full Length Ref. Sequences:
Indicates the references to multiple sequences in RefSeq. The field contains the ID and description for each entry, and there can be multiple entries per probeSet.
Example 3: Sample preparation, processing and data analysis
Method 1:
Microanay analyses were performed utilizing the GeneChip® System (Affymetrix, Santa Clara, USA). Hybridization target preparations were performed according to recommended protocols (Affymetrix Technical Manual). In detail, at time of diagnosis, mononuclear cells were purified by Ficoll-Hypaque density centrifugation. They had been lysed immediately in RLT buffer (Qiagen, Hilden, Germany), frozen, and stored at -80°C from 1 week to 38 months. For gene expression profiling cell lysates of the leukemia samples were thawed, homogenized (QIAshredder, Qiagen), and total RNA was extracted (RNeasy Mini
Kit, Qiagen). Subsequently, 5-10 μg total RNA isolated from 1 x 107 cells was used as starting material for cDNA synthesis with oligo [(dT)24T7promotor]65 primer (cDNA Synthesis System, Roche Applied Science, Mannheim, Germany). cDNA products were purified by phenol/chlorophorm/IAA extraction (Ambion, Austin, USA) and acetate/ethanol-precipitated overnight. For detection of the hybridized target nucleic acid biotin-labeled ribonucleotides were incorporated during the following in vitro transcription reaction (Enzo BioAnay HighYield RNA Transcript Labeling Kit, Enzo Diagnostics). After quantification by spectrophotometric measurements and 260/280 absorbance values assessment for quality control of the purified cRNA (RNeasy Mini Kit, Qiagen), 15 μg cRNA was fragmented by alkaline treatment (200 mM Tris-acetate, pH 8.2/500 mM potassium acetate/150 mM magnesium acetate) and added to the hybridization cocktail sufficient for five hybridizations on standard GeneChip microanays (300 μl final volume). Washing and staining of the probe anays was performed according to the recommended Fluidics Station protocol (EukGE-WS2v4). Affymetrix Microanay
Suite software (version 5.0.1) extracted fluorescence signal intensities from each feature on the microanays as detected by confocal laser scanning according to the manufacturer's recommendations.
Expression analysis quality assessment parameters included visiual anay inspection of the scanned image for the presence of image artifacts and conect grid alignment for the identification of distinct probe cells as well as both low 375' ratio of housekeeping controls (mean: 1.90 for GAPDH) and high percentage of detection calls (mean: 46.3% present called genes). The 3' to 5' ratio of GAPDH probesets can be used to assess RNA sample and assay quality. Signal values of the 3' probe sets for GAPDH are compared to the Signal values of the conesponding
5' probe set. The ratio of the 3' probe set to the 5' probe set is generally no more than 3.0. A high 3' to 5' ratio may indicate degraded RNA or inefficient synthesis of ds cDNA or biotinylated cRNA (GeneChip® Expression Analysis Technical Manual, www.affymetrix.com). Detection calls are used to determine whether the transcript of a gene is detected (present) or undetected (absent) and were calculated using default parameters of the Microanay Analysis Suite MAS 5.0 software package.
Method 2: Bone manow (BM) aspirates are taken at the time of the initial diagnostic biopsy and remaining material is immediately lysed in RLT buffer (Qiagen), frozen and stored at -80 C until preparation for gene expression analysis. For microanay analysis the GeneChip System (Affymetrix, Santa Clara, CA, USA) is used. The targets for GeneChip analysis are prepared according to the cunent Expression Analysis. Briefly, frozen lysates of the leukemia samples are thawed, homogenized
(QIAshredder, Qiagen) and total RNA extracted (RNeasy Mini Kit, Qiagen) .Normally 10 ug total RNA isolated from 1 x 107 cells is used as starting material in the subsequent cDNA-Synthesis using Oligo-dT-T7-Promotor Primer (cDNA synthesis Kit, Roche Molecular Biochemicals). The cDNA is purified by phenol-chlorophorm extraction and precipitated with 100% Ethanol over night. For detection of the hybridized target nucleic acid biotin-labeled ribonucleotides are incorporated during the in vitro transcription reaction (Enzo® BioAnay™ HighYield™ RNA Transcript Labeling Kit, ENZO). After quantification of the purified cRNA (RNeasy Mini Kit, Qiagen), 15 ug are fragmented by alkaline treatment (200 mM Tris-acetate, pH 8.2, 500 mM potassium acetate, 150 mM magnesium acetate) and added to the hybridization cocktail sufficient for 5 hybridizations on standard GeneChip microanays. Before expression profiling Test3 Probe Anays (Affymetrix) are chosen for monitoring of the integrity of the cRNA. Only labeled cRNA-cocktails which showed a ratio of the messured intensity of the 3' to the 5' end of the GAPDH gene less than 3.0 are selected for subsequent hybridization on HG-U133 probe anays (Affymetrix). Washing and staining the Probe anays is performed as described (siehe Affymetrix-Original- Literatur (LOCKHART und LIPSHUTZ). The Affymetrix software (Microanay Suite, Version 4.0.1) extracted fluorescence intensities from each element on the anays as detected by confocal laser scanning according to the manufacturers recommendations .
Table 1
Figure imgf000033_0001
Figure imgf000034_0001
Tables 2 and 3
Table 2
One-Versus-AII (OVA)
Figure imgf000035_0001
Tables 2 and 3
Figure imgf000036_0001
Tables 2 and 3
Figure imgf000037_0001
Figure imgf000038_0001
Figure imgf000039_0001
Figure imgf000040_0001
Figure imgf000041_0001
Figure imgf000042_0001
Table 3
3. All-Pairs (AP) 3.1 M4eo versus PTD
# affy id HUGO name fc P q stn t Map Location 1 235753_at -8.40 1.24E-10 4.49E-07 -2.15 -11.39 2 206847_s_at HOXA7 -5.18 2.73E-11 2.32E-07 -1.81 -10.98 7p15-p14 3 201497_x_at MYH11 18.86 2.02E-10 6.27E-07 2.05 10.66 16p13.13- p13.12 4 213908_at -7.48 4.93E-10 1.14E-06 -1.78 -10.15 5 213147_at HOXA10 -5.00 9.04E-11 4.25E-07 -1.60 -9.96 7p15-p14 6 235359_at 3.64 1.33E-11 1.95E-07 1.48 9.72 7214651_s_at HOXA9 -17.28 3.1 OE-09 3.62E-06 -1.80 -9.54 7p15-p14 8 201496_x_at MYH11 5.22 1.00E-10 4.25E-07 1.49 9.47 16p13.13- p13.12 9 20O953_s_at CCND2 2.28 1.53E-11 1.95E-07 1.40 9.35 12p13 10 209406_at BAG2 -5.34 3.13E-09 3.62E-06 -1.62 -9.21 6p12.3- p11.2 11 20O951_s_at CCND2 3.03 6.00E-11 3.81 E-07 1.33 8.89 12p13 I2 213150_at HOXA10 -7.80 1.12E-08 9.12E-06 -1.55 -8.67 7p15-p14 I3 21 963_s_at NGFRAP1 -13.60 1.45E-08 1.02E-05 -1.52 -8.52 Xq22.1 42 Tables 2 and 3 14 202746_at ITM2A 3.65 2.22E-10 6.27E-07 1.27 8.48 Xq13.3- Xq21.2 15 202747_s_at ITM2A 3.99 2.69E-10 6.85E-07 1.26 8.40 Xq13.3- Xq21.2 16 227533_at -2.80 1.75E-09 2.62E-06 -1.32 -8.40 17 226352_at -7.39 2.41 E-08 1.32E-05 -1.51 -8.35 18 205330_at MN1 8.19 1.15E-08 9.12E-06 1.43 8.29 22q12.1 19 226944_at HTRA3 -4.12 6.84E-09 5.79E-06 -1.35 -8.274p16.1 20 209365_s_at ECM1 2.54 6.72E-10 1.31 E-06 1.25 8.24 1q21 21 223385_at CYP2S1 2.26 1.16E-09 1.84E-06 1.24 8.14 19q13.1 22 201005_at CD9 6.33 2.35E-09 3.02E-06 1.26 8.11 12p13.3 23 205600_x_at HOXB5 -2.96 3.92E-08 1.84E-05 -1.37 -7.92 17q21.3 24 218214_at FLJ11773 1.96 6.32E-10 1.31 E-06 1.16 7.87 12q13.13 25 205830_at CLGN -7.01 5.00E-08 2.27E-05 -1.38 -7.87 4q28.3- q31.1 26 220591_s_at FLJ22843 2.52 1.54E-08 1.03E-05 1.27 7.82 Xp11.3 27 211926_s_at MYH9 1.88 9.38E-10 1.59E-06 1.15 7.80 22q13.1 28 217849_s_at CDC42BPB 4.70 8.99E-10 1.59E-06 1.15 7.79 14q32.3 29 224772_at NAV1 2.53 4.15E-09 4.58E-06 1.19 7.77 30 225055_at DKFZp667M2411 -3.67 1.76E-08 1.08E-05 -1.24 -7.75 17q11.2 31 209905_at H0XA9 -48.57 1.40E-07 4.33E-05 -1.56 -7.72 7p15-p14 32 243010_at MSI2 -3.13 8.47E-08 3.12E-05 -1.37 -7.70 17q23.1 33 241985_at FLJ37870 -7.23 7.12E-08 2.72E-05 -1.34 -7.69 5q13.3 34 227224_at FLJ25604 -4.72 3.63E-08 1.77E-05 -1.25 -7.62 1q24.2 35 212358_at CLIPR-59 14.29 9.72E-08 3.43E-05 1.48 7.61 19q13.12 36 208033_s_at ATBF1 3.24 5.57E-09 5.06E-06 1.15 7.57 16q22.3- q23.1 37 225346_at LOC80298 -2.05 1.51 E-08 1.03E-05 -1.18 -7.54 12q24.1 38 209190_s_at DIAPH1 1.99 1.89E-09 2.67E-06 1.11 7.54 5q31 39 34210_at CDW52 3.20 2.37E-09 3.02E-06 1.11 7.49 1p36 40 21O139_s_at PMP22 5.29 1.35E-08 9.81 E-06 1.16 7.48 17p12- p11.2 41 223044_at SLC11A3 -9.10 1.27E-07 4.04E-05 -1.31 -7.46 2q32 42 241 525_at LOC200772 45.22 1.44E-07 4.36E-05 1.44 7.43 2q37.3 43 224998_at CKLFSF4 -2.08 5.48E-08 2.40E-05 -1.21 -7.42 16q21 44 21O150_s_at LAMA5 2.45 4.44E-09 4.71 E-06 1.10 7.40 20q13.2- q13.3 45 230896_at -19.18 2.83E-07 6.17E-05 -1.47 -7.37 46 208873_s_at DP1 3.10 1.79E-08 1.08E-05 1.13 7.35 5q22-q23 47 222786_at C4S-2 -3.35 1.76E-07 4.82E-05 -1.29 -7.32 7p22 48 20O984_s_at CD59 -4.41 2.01 E-07 5.16E-05 -1.29 -7.29 11p13 49 201389_at ITGA5 2.13 6.03E-08 2.54E-05 1.19 7.28 12q11- q13 50 218418 s at KIAA1518 -2.74 1.02E-07 3.49E-05 -1.21 -7.28 19p13.13
3.2 M4eo versus inv3
# affy id HUGO name fc p q stn t Map Location 1 203949 at MPO 4.74 1.72E-13 4.54E-09 2.41 14.22 17q23.1 43 Tables 2 and 3 2 203948_s_at MPO 5.13 2.36E-12 2.08E-08 1.89 11.46 17q23.1 3 205382_s_at DF 5.65 1.05E-12 1.38E-08 1.83 11.19 19p13.3 4 201497_x_at MYH11 18.46 2.05E-10 7.07E-07 2.06 10.65 16p13.13- p13.12 5 224841_x_at -1.69 2.14E-10 7.07E-07 -1.76 -10.33 6 224741_x_at -1.69 3.09E-10 9.08E-07 -1.76 -10.28 7 209365_s_at ECM1 3.28 3.37E-11 2.23E-07 1.54 9.53 1q21 8 210755_at HGF 6.18 6.96E-10 1.84E-06 1.65 9.44 7q21.1 9 228497_at FLIPT1 -3.11 8.82E-09 1.17E-05 -1.63 -9.19 1p13.1
10 205718_at ITGB7 3.07 1.91 E-10 7.07E-07 1.44 8.88 12q13.13
1 1 205131_x_at SCGF 4.37 1.79E-10 7.07E-07 1.40 8.73 19q13.3
12 217963_s_at NGFRAP1 -20.39 5.19E-07 1.67E-04 -1.88 -8.49 Xq22.1
13 201496_x_at MYH11 3.64 1.43E-09 3.16E-06 1.40 8.45 16p13.13- p13.12
14 222862_s_at AK5 40.65 3.10E-08 2.93E-05 1.61 8.14 1p31
15 236646_at FLJ31166 3.02 9.63E-10 2.31 E-06 1.30 8.12 12p13.31
16 226197_at 2.75 2.51 E-09 4.46E-06 1.31 8.04
17 203074_at ANXA8 1.80 2.08E-09 4.22E-06 1.30 8.04 10q11.2
18 243244_at 3.90 2.53E-09 4.46E-06 1.29 7.95
19 202605_at GUSB 2.22 4.26E-08 3.47E-05 1.30 7.70 7q21.11
20 212358_at CLIPR-59 15.49 8.58E-08 5.04E-05 1.46 7.63 19q13.12
21 201360_at CST3 3.63 4.80E-09 7.94E-06 1.22 7.62 20p11.21
22 226697_at LOC92689 2.52 6.69E-09 .1.04E-05 1.22 7.58 4p14
23 201462_at KIAA0193 -5.29 3.06E-07 1.13E-04 -1.37 -7.57 7p14.3- p14.1
24 241525_at LOC200772 55.36 1.35E-07 6.48E-05 1.47 7.46 2q37.3
25 210783_x_at SCGF 4.12 8.13E-09 1.13E-05 1.20 7.46 19q13.3
26 231736_x_at MGST1 3.57 7.41 E-09 1.09E-05 1.19 7.44 12p12.3- p12.1
27 207961_x_at MYH11 15.00 1.40E-07 6.63E-05 1.43 7.42 16p13.13- p13.12
28 224441_s_at MGC14793 -3.13 8.20E-08 5.04E-05 -1.24 -7.37 6q16.3
29 205076_s_at CRA 4.21 4.89E-08 3.77E-05 1.24 7.34 1q12-q21
30 210997_at HGF 17.75 1.55E-07 6.94E-05 1.38 7.34 7q21.1
31 209975_at CYP2E1 3.46 4.33E-08 3.47E-05 1.22 7.30 10q24.3- qter
32 224918_x_at MGST1 3.27 1.58E-08 1.90E-05 1.18 7.29 12p12.3- p12.1
33 201069_at MMP2 2.83 1.26E-08 1.59E-05 1.17 7.28 16q13- q21
34 202828_s_at MMP14 5.47 1.26E-07 6.34E-05 1.29 7.25 14q11- q12
35 211709_s_at SCGF 3.22 48E-08 3.08E-05 1.18 7.24 19q13.3
36 202283_at SERPINF1 4.68 61 E-08 3.08E-05 1.18 7.18 17p13.1
37 200852_x_at GNB2 2.10 31 E-08 2.65E-05 1.15 7.16 7q22
38 201688_s_at TPD52 -3.31 77E-07 2.23E-04 -1.30 -7.14 8q21
39 219308_s_at AK5 5.75 20E-07 9.06E-05 1.32 7.14 1p31
40 239814_at 2.34 50E-08 2.75E-05 1.14 7.12
41 200985_s_at CD59 -6.95 61 E-06 5.15E-04 -1.42 -7.0911p13
42 242621_at FLJ32468 1.47 87E-08 2.81 E-05 1.14 7.087q22.1
43 202185_at PLOD3 1.78 78E-08 2.81 E-05 1.14 7.077q22
44 223136 at AIG-1 -5.06 07E-07 2.45E-04 -1.28 -7.056q24.1 44 Tables 2 and 3
45 223091_x_at GL004 -1.53 1.27E-07 6.34E-05 -1.17 -7.04 2q36.3
46 223354_x_at GL004 -1.62 2.88E-07 1.09E-04 -1.21 -7.04 2q36.3
47 214797_s_at PCTK3 -2.39 4.15E-07 1.44E-04 -1.22 -7.03 1q31-q:
48 214558_at GPR12 1.53 4.99E-08 3.77E-05 1.14 7.01 13q12
49 229309_at 4.49 6.27E-08 4.25E-05 1.15 7.01
50 205859_at LY86 3.30 2.78E-08 2.81 E-05 1.12 7.01 6p24.3
3.3 M4eo versus t(15;17) affy id HUGO name fc P q stn 1 Map Location 1 211990_at HLA-DPA1 12.88 7.26E-18 1.92E-13 3.35 20.08 6p21.3 2 214450_at CTSW -8.03 6.77E-13 7.14E-10 -3.05 -15.96 11q13.1 3 38487_at STAB1 -8.03 2.37E-12 1.95E-09 -3.01 -15.25 3p21.31 4 221004_s_at ITM2C -5.22 1.41 E-13 3.01 E-10 -2.58 -15.04 2q37 5 204661_at CDW52 33.75 1.67E-13 3.15E-10 2.69 14.74 1p36 6 200654_at P4HB -2.30 1.92E-15 1.27E-11 -2.31 -14.63 17q25 7 203535_at S100A9 9.01 7.53E-16 6.62E-12 2.24 14.32 1q21 8 217478_s_at HLA-DMA 7.63 2.80E-14 8.72E-11 2.35 14.21 6p21.3 9 209732_at CLECSF2 30.47 5.76E-13 6.61E-10 2.71 14.20 12p13- p12 10 34210_at CDW52 43.85 7.27E-13 7.14E-10 2.58 13.90 1p36 11 238022_at -8.74 2.99E-12 2.25E-09 -2.41 -13.63 12 209619_at CD74 5.65 3.24E-16 4.28E-12 2.06 13.52 5q32 13 201923_at PRDX4 7.22 7.48E-14 1.79E-10 2.16 13.28 Xp22.13 14 205624_at CPA3 -9.54 1.00E-11 6.01 E-09 -2.41 -13.24 3q21-q25 15 204563_at SELL 9.35 7.30E-13 7.14E-10 2.25 13.07 1q23-q25 16 200931_s_at VCL 3.96 1.06E-14 5.62E-11 2.01 12.90 10q22.1- q23 17 231310_at 4.74 2.97E-14 8.72E-11 2.04 12.89 18 209312_x_at HLA-DRB1 8.89 3.15E-13 4.37E-10 2.06 12.62 6p21.3 19 208306_x_at HLA-DRB4 9.65 5.23E-13 6.43E-10 2.08 12.60 6p21.3
20 238365_s_at -10.74 1.01 E-10 3.36E-08 -2.50 -12.45
21 208891_at DUSP6 7.70 2.11E-14 8.72E-11 1.93 12.44 12q22- q23
22 212953_x_at CALR -2.84 2.97E-14 8.72E-11 -1.91 -12.34 19p13.3- p13.2
23 204670_x_at HLA-DRB5 6.79 3.94E-14 1.04E-10 1.91 12.25 6p21.3
24 205718_at ITGB7 6.61 6.63E-13 7.14E-10 1.97 12.10 12q13.13
25 205453_at HOXB2 11.16 1.03E-11 6.03E-09 2.13 11.95 17q21- q22
26 205663_at PCBP3 -4.69 1.37E-11 7.52E-09 -2.01 -11.85 21 q22.3
27 232617_at CTSS 8.88 1.90E-11 9.29E-09 2.15 11.78 1q21
28 207375_s_at IL15RA 4.80 1.48E-13 3.01 E-10 1.84 11.77 10p15- p14
29 224583_at COTL1 5.58 3.11 E-13 4.37E-10 1.86 11.77 16q23.3
30 221059_s_at CHST6 6.80 4.13E-12 2.80E-09 1.95 11.67 16q22
31 233072_at KIAA1857 -7.47 2.04E-10 5.49E-08 -2.19 -11.60 9q34 32 229168_at DKFZp434K0621 -6.73 3.74E-10 8.88E-08 -2.36 -11.59 5q35.3
33 208982_at PECAM1 4.84 2.17E-12 1.85E-09 1.88 11.55 17q23 45 Tables 2 and 3 34 224839_s_at GPT2 -9.02 4.23E-11 1.75E-08 -1.95 -11.41 16q12.1 35 202803_s_at ITGB2 5.43 5.36E-13 6.43E-10 1.72 11.07 21 q22.3 36 223280_x_at MS4A6A 24.98 9.94E-11 3.36E-08 2.11 11.04 11q12.1 37 20 496_x_at MYH11 10.61 1.13E-11 6.47E-09 1.81 10.98 16p13.13- p13.12 38 21 991_s_at HLA-DPA1 25.17 9.82E-11 3.36E-08 2.05 10.97 6p21.3 39 204150_at STAB1 -9.71 1.08E-09 2.11 E-07 -2.26 -10.94 3p21.31 40 208689_s_at RPN2 -1.75 1.91E-13 3.36E-10 -1.66 -10.90 20q12- q13.1 41 220798_x_at FLJ 11535 -3.81 7.69E-11 2.82E-08 -1.84 -10.89 19p13.3 42 201497_x_at MYH11 28.44 1.48E-10 4.48E-08 2.16 10.88 16p13.13- p13.12 43 202917_s_at S100A8 3.19 3.79E-13 5.01E-10 1.66 10.85 1q21 44 241742_at PRAM-1 11.60 1.23E-10 3.81 E-08 1.97 10.76 19p13.2 45 228046_at LOG 152485 3.03 5.49E-12 3.54E-09 1.72 10.76 4q31.1 46 226878_at 4.19 1.90E-11 9.29E-09 1.77 10.75 47 238604_at 3.63 2.30E-13 3.79E-10 1.62 10.71 48 213779_at LOC 129080 -6.64 9.66E-10 1.96E-07 -2.04 -10.68 22q12.1 49 224356_x_at MS4A6A 25.23 2.22E-10 5.74E-08 2.06 10.62 11q12.1 50 217897 at FXYD6 3.03 3.34E-11 1.44E-08 1.77 10.62 11 q23.3
3.4 M4eo versus t(821)
# affy id HUGO name fc P stn Map Location 1 207075_at CIAS1 6.60 1.43E 12 1.58E-08 2.19 12.64 1q44 2 208890_s_at PLXNB2 5.22 2.59E 13 7.61 E-09 1.97 12.1622q13.33 3 205453_at HOXB2 12.65 7.66E •12 3.63E-08 2.15 12.0717q21- q22 4 205419_at EBI2 7.98 2.83E •12 2.35E-08 2.05 12.0313q32.2 5 205718_at ITGB7 6.53 4.59E 13 7.61 E-09 1.89 11.7512q13.13 6 224764_at ARHGAP10 8.90 1.13E ■11 4.17E-08 2.01 11.58 10 7 218795_at ACP6 -4.56 4.74E •11 1.12E-07 -1.87 11.101q21 8 201497_x_at MYH11 26.30 1.55E 10 2.09E-07 2.14 10.8516p13.13- p13.12 9201496_x_at MYH11 9.04 1.66E -11 5.52E-08 1.78 10.7416p13.13- p13.12 10 20O665_s_at SPARC 4.57 5.30E 12 2.93E-08 1.71 10.655q31.3- q32 11 224049_at KCNK17 4.59 1.14E 10 1.80E-07 1.91 10.656p21.1 12 224724_at SULF2 27.22 4.07E 10 3.97E-07 1.99 10.2920q12- 13.2 13 218236_s_at PRKCN 4.94 4.24E 12 2.81 E-08 1.60 10.202p21 14 20 425_at ALDH2 7.88 2.04E 10 2.42E-07 1.71 9.9812q24.2 15 203320_at LNK 3.26 9.11 E 11 1.71 E-07 1.62 9.8312q24 16 201944_at HEXB 2.27 3.74E 11 9.55E-08 1.57 9.805q13 17 201360_at CST3 5.61 9.57E 11 1.71 E-07 1.59 9.7220p11.21 18 209365_s_at ECM1 3.24 2.77E 11 7.66E-08 1.52 9.611q21 19 201887_at IL13RA1 4.89 3.33E 10 3.57E-07 1.62 9.59 Xq24 20 220974_x_at BA108L7.2 5.51 2.19E 10 2.51 E-07 1.57 9.5210q24.31 21 201596_x_at KRT18 7.84 1.81 E 10 2.24E-07 1.56 9.5212q13 46 Tables 2 and 3
22 221841_s_at 4.33 2.08E-11 6.27E-08 1.48 9.48
23 238604_at 3.14 1.08E-11 4.17E-08 1.45 9.41
24 202670_at MAP2K1 3.54 5.66E-10 5.22E-07 1.58 9.36 15q22.1- q22.33
25 210314_x_at TNFSF13 4.72 3.31 E-10 3.57E-07 1.52 9.25 17p13.1
26 209500_x_at TNFSF13 3.94 5.43E-10 5. 5E-07 1.54 9.24 17p13.1
27 235359_at 2.92 1.82E-10 2.24E-07 1.46 9.12
28 223249_at CLD 12 3.41 1.57E-10 2.09E-07 1.43 9.00 7q21
29 201739_at SGK 4.50 5.38E-11 1.19E-07 1.39 8.97 6q23
30 229309_at 11.01 3.92E-09 2.32E-06 1.64 8.96
31 206940_s_at POU4F1 -40.05 7.47E-08 1.80E-05 -2.03 -8.95 13q21.1- q22
32 218217_at RISC 3.30 1.43E-10 2.06E-07 1.41 8.94 17q23.1
33 208683_at CAPN2 3.27 9.79E-11 1.71 E-07 1.38 8.88 1q41-q42
34 226818_at LOC219972 10.92 2.55E-09 1.77E-06 1.53 8.85 11q12.1
35 240572_s_at 3.25 1.23E-10 1.86E-07 1.37 8.80
36 212459_x_at SUCLG2 3.68 8.62E-11 1.71 E-07 1.35 8.76 3p14.2
37 229383_at 4.93 3.73E-09 2.27E-06 1.51 8.71
38 205859_at LY86 3.62 1.25E-09 1.04E-06 1.42 8.67 6p24.3
39 225602_at C9orf19 2.80 1.09E-10 1.80E-07 1.34 8.67 9p13-p12
40 211341_at POU4F1 1.28E-07 2.73E-05 -2.00 -8.63 13q21.1- 165.76 q22
41 203329_at PTPRM 6.43 4.01 E-09 2.33E-06 1.48 8.61 18p11.2
42 205330_at MN1 9.71 9.34E-09 4.25E-06 1.60 8.60 22q12.1
43 204057_at ICSBP1 4.44 4.46E-09 2.40E-06 1.47 8.57 16q24.1
44 236738_at 6.32 1.86E-09 1.40E-06 1.39 8.50
45 21 084_x_at PRKCN 4.65 3.64E-10 3.67E-07 1.31 8.41 2p21
46 217849_s_at CDC42BPB 4.67 3.44E-10 3.57E-07 1.30 8.39 14q32.3
47 208033_s_at ATBF1 3.91 1.05E-09 9.16E-07 1.31 8.30 16q22.3- q23.1
48 205076_s_at CRA 5.74 1.33E-08 5.44E-06 1.47 8.27 1q12-q21
49 228827_at 2.39E-07 4.54E-05 -1.91 -8.25 103.82
50 226841 at L0C219972 12.37 1.88E-08 6.92E-06 1.51 8.24 11q12.1
3.5 M4eo versus tMLL affy id HUGO name fc P q J stn I i Map Location 1 213737_x_at -3.81 2.63E-16 7.45E-12 -2.33 -15.21 2 200665_s_at SPARC 16.92 2.60E-13 1.47E-09 2.28 13.71 5q31.3- q32 3 214651_s_at HOXA9 -24.73 4.60E-14 3.26E-10 -2.26 -13.54 7p15-p14 4 200953_s_at CCND2 4.36 1.06E-15 1.50E-11 1.96 13.49 12p13 5 202746_at ITM2A 15.99 1.64E-12 4.65E-09 2.15 12.76 Xq13.3- Xq21.2 6 202747_s_at ITM2A 16.03 3.21 E-12 8.28E-09 2.02 12.22 Xq13.3- Xq21.2 7 200951_s_at CCND2 5.31 4.09E-13 1.66E-09 1.80 11.91 12p13 8 231310_at 4.76 7.45E-15 7.04E-11 1.67 11.82 9 202551_s_at CRIM1 4.27 3.61 E-13 1.66E-09 1.61 11.06 2p21 47 Tables 2 and 3
10 227567_at -5.39 7.34E-13 2.60E-09 -1.62 -10.92
11 201497_x_at MYH11 26.26 1.56E-10 1.30E-07 2.13 10.85 16p13.13- p13.12
12 205453_at HOXB2 7.94 5.98E-12 1.30E-08 1.65 10.82 17q21- q22
13 224049_at KCNK17 4.81 8.48E-11 8.90E-08 1.85 10.77 6p21.1
14 235753_at -13.72 2.38E-11 3.96E-08 -1.85 -10.59
15 201496_x_at MYH11 6.89 5.88E-11 7.25E-08 1.72 10.56 16p13.13- p13.12
16 212667_at SPARC 8.11 5.29E-11 6.97E-08 1.64 10.33 5q31.3- q32
17 206847_s_at HOXA7 -6.82 1.92E-11 3.41 E-08 -1.61 -10.23 7p15-p14
18 229215_at ASCL2 -10.76 3.29E-11 4.91 E-08 -1.63 -10.12 11p15.5
19 209905_at HOXA9 -81.11 8.12E-11 8.85E-08 -1.80 -10.06 7p15-p14
20 202931_x_at BIN1 3.10 1.12E-12 3.53E-09 1.42 10.04 2q14
21 213147_at HOXA10 -6.16 1.50E-11 2.84E-08 -1.51 -9.96 7p15-p14
22 201830_s_at NET1 4.25 1.11 E-10 1.12E-07 1.50 9.70 10p15
23 226517_at BCAT1 10.34 5.88E-10 3.33E-07 1.61 9.63 12pter- q12
24 213150_at HOXA10 -10.83 1.56E-10 1.30E-07 -1.57 -9.57 7p15-p14
25 213908_at -15.52 3.82E-10 2.40E-07 -1.60 -9.31
26 204082_at PBX3 -5.53 3.07E-10 2.12E-07 -1.54 -9.31 9q33-q34
27 228058_at LOG 124220 6.00 6.45E-12 1.31 E-08 1.29 9.24 16p13.3
28 203949_at MPO 3.13 3.59E-11 5.08E-08 1.33 9.17 17q23.1
29 242738_s_at 2.48 2.90E-10 2.06E-07 1.40 9.16
30 225831_at LOC148894 3.66 1.72E-10 1.37E-07 1.37 9.16 1p36.11
31 224952_at DKFZP564D166 -3.41 4.56E-12 1.08E-08 -1.27 -9.14 17q23.3
32 202370_s_at CBFB -3.09 2.04E-10 1.49E-07 -1.41 -9.12 16q22.1
33 205330_at MN1 17.21 4.19E-09 1.40E-06 1.73 9.08 22q12.1
34 223471_at RAB3IP -3.52 7.55E-11 8.56E-08 -1.32 -9.03
35 223385_at CYP2S1 2.42 3.14E-10 2.12E-07 1.36 9.02 19q13.1
36 210139_s_at PMP22 9.18 3.17E-09 1.18E-06 1.54 8.97 17p12- p11.2
37 201029_s_at CD99 1.88 3.17E-11 4.91 E-08 1.26 8.91 Xp22.32
38 226137_at 3.72 1.92E-09 8.26E-07 1.43 8.86
39 218966_at MY05C 3.05 2.27E-09 9.48E-07 1.41 8.76 15q21
40 224772_at NAV1 2.83 8.86E-10 4.74E-07 1.34 8.76
41 203733_at MYLE -3.28 1.29E-10 1.26E-07 -1.27 -8.75 16p13.2
42 203329_at PTPRM 6.00 6.69E-09 1.95E-06 1.52 8.68 18p11.2
43 211012_s_at PML 2.73 5.41 E-11 6.97E-08 1.22 8.68 15q22
44 202265_at BMI1 -3.09 3.65E-10 2.40E-07 -1.30 -8.66 10p11.23
45 214452_at BCAT1 4.20 1.00E-09 4.89E-07 1.31 8.63 12pter- q12
46 242686_at 2.41 2.36E-09 9.69E-07 1.36 8.62
47 212771_at LOC221061 5.21 1.07E-08 2.68E-06 1.58 8.58 10p13
48 200602_at APP 6.12 1.47E-10 1.30E-07 1.22 8.57 21q21.3
49 228496_s_at CRIM1 2.79 1.54E-10 1.30E-07 1.21 8.52 2p21
50 210006_at DKFZP5640243 -2.19 7.82E-10 4.34E-07 -1.29 -8.49 3p21.1
3.6 PTD versus inv3 48 Tables 2 and 3 affy id HUGO name fc P q stn t Map Location 1 229116_at 8.14 5.54E-07 1.82E-03 1.33 6.95 2 235753_at 2.97 5.69E-08 1.40E-03 1.13 6.87 3 2056O0_x_at HOXB5 2.38 3.90E-07 1.82E-03 1.14 6.60 17q21.3 4 214643_x_at BIN1 -2.90 2.16E-06 2.51 E-03 -1.15 -6.43 2q14 5 205382_s_at DF 4.56 1.23E-06 2.02E-03 1.10 6.28 19p13.3 6 209679_s_at LOC57228 -3.63 6.67E-06 5.48E-03 -1.19 -6.27 12q13.12 7 228161_at RAB32 1.67 5.28E-07 1.82E-03 1.05 6.26 6q24.2 8 226697_at LOC92689 2.24 4.73E-07 1.82E-03 1.04 6.25 4p14 9 211084_x_at PRKCN -2.18 8.67E-07 2.02E-03 -1.05 -6.24 2p21
10 2131 10_s_at COL4A5 18.16 3.89E-06 3.68E-03 1.26 6.17 Xq22
11 224918_x_at MGST1 3.11 5.28E-07 1.82E-03 1.01 6.13 12p12.3- p12.1
12 231736_x_at MGST1 3.26 5.91 E-07 1.82E-03 1.01 6.11 12p12.3- p12.1
13 215016_x_at BPAG1 3.91 5.81 E-07 1.82E-03 1.00 6.10 6p12-p11
14 226789_at 2.44 1.21 E-06 2.02E-03 1.03 6.08
15 233893_s_at KIAA1530 1.52 7.27E-07 1.99E-03 1.00 6.04 4p16.3
16 232250_at KIAA1257 3.80 2.55E-06 2.51 E-03 1.05 5.99 3q21.3
17 218552_at FLJ 10948 2.00 9.91 E-07 2.02E-03 0.99 5.98 1p32.3
18 226197_at 2.36 1.63E-06 2.34E-03 1.00 5.94
19 206847_s_at HOXA7 2.13 1.13E-06 2.02E-03 0.97 5.88 7p15-p14
20 2187O9_s_at C20orf9 1.60 1.18E-06 2.02E-03 0.97 5.87
21 236892_s_at 6.01 8.37E-06 6.10E-03 1.11 5.78
22 212254_s_at BPAG1 3.37 1.51 E-06 2.32E-03 0.95 5.78 6p12-p11
23 2094O6_at BAG2 2.41 1.99E-06 2.51 E-03 0.95 5.75 6p12.3- p11.2
24 225464_at C14orf31 2.28 1.71 E-06 2.34E-03 0.94 5.74 14q21.3
25 228252_at PIF1 2.29 2.21 E-06 2.51 E-03 0.95 5.73 15q22.1
26 205767_at EREG 11.02 9.66E-06 6.10E-03 1.10 5.72 4q21.1
27 205830_at CLGN 3.48 2.41 E-06 2.51 E-03 0.94 5.68 4q28.3- q31.1
28 205514_at FLJ11191 -2.72 1.42E-05 7.13E-03 -1.03 -5.67 19q13.41
29 240151_at 2.28 2.31 E-06 2.51 E-03 0.93 5.66
30 205330_at MN1 -7.45 4.85E-05 1.09E-02 -1.23 -5.65 22q12.1
31 214651_s_at HOXA9 3.07 2.52E-06 2.51 E-03 0.93 5.63 7p15-p14
32 201829_at NET1 -2.38 2.74E-05 8.76E-03 -1.03 -5.52 10p15
33 2043O1_at KIAA0711 4.79 1.26E-05 7.12E-03 0.99 5.48 8p23.2
34 242621_at FLJ32468 1.54 9.56E-06 6.10E-03 0.95 5.45 7q22.1
35 230051_at -2.32 2.38E-05 8.38E-03 -0.98 -5.43
36 244297_at FLJ35740 3.45 1.56E-05 7.13E-03 0.98 5.40 9p12
37 202232_s_at GA17 -1.60 6.52E-06 5.48E-03 -0.90 -5.39 11p13
38 213147_at HOXA10 2.19 5.23E-06 4.77E-03 0.89 5.39 7p15-p14
39 2099O5_at HOXA9 4.12 8.60E-06 6.10E-03 0.91 5.36 7p15-p14
40 2056O1_s_at HOXB5 2.42 5.97E-06 5.25E-03 0.88 5.36 17q21.3
41 232424_at PRDM16 5.47 8.84E-06 6.10E-03 0.90 5.35 1p36.23- p33
42 213150_at HOXA10 2.61 7.01 E-06 5.57E-03 0.88 5.33 7p15-p14
43 239791 at 5.52 1.96E-05 7.90E-03 0.98 5.33 49 Tables 2 and 3 44 214684_at MEF2A -1.80 1.23E-05 7.12E-03 -0.90 -5.32 15q26 45 202600_s_at NRIP1 -3.90 8.68E-05 1.23E-02 -1.13 -5.31 21q11.2 46 203462_x_at EIF3S9 1.73 7.66E-06 5.89E-03 0.87 5.29 7p22.3 47 223463_at RAB23 2.75 1.33E-05 7.13E-03 0.91 5.29 6p11.2- p12.3 48 216035_x_at TCF7L2 -2.37 4.09E-05 1.08E-02 -0.97 -5.28 10q25.3 49 206725_x_at BMP1 1.74 1.60E-05 7.13E-03 0.91 5.26 8p21 50 222755 s at KIAA1416 1.70 1.04E-05 6.22E-03 0.88 5.25 8q12.1
3.7 PTD versus t(15; 17)
# affy id HUGO name fc P q stn t Map Location 1 214450_at CTSW -8.49 5.28E-14 2.10E-10 -2.60 -15.24 11q13.1 2 221004_s_at ITM2C -5.89 3.54E-15 3.51 E-11 -2.19 -13.79 2q37 3 38487_at STAB1 -6.66 2.22E-13 7.33E-10 -2.30 -13.71 3p21.31 4 212953_x_at CALR -3.25 6.25E-15 4.13E-11 -2.14 -13.47 19p13.3- p13.2 5 214789_x_at SRP46 4.03 1.12E-15 2.22E-11 2.07 13.30 11q22 6 213147_at HOXA10 19.18 1.60E-11 1.80E-08 2.44 12.72 7p15-p14 7 200654_at P4HB -2.49 1.62E-14 8.06E-11 -1.82 -11.76 17q25 8 206847_s_at HOXA7 6.88 9.35E-12 1.33E-08 2.00 11.70 7p15-p14 9 235753_at 10.05 9.19E-11 6.52E-08 2.29 11.69 10 233072_at KIAA1857 -7.46 8.29E-11 6.19E-08 -1.96 -11.21 9q34 11 212509_s_at -6.36 2.03E-10 1.30E-07 -2.05 -11.21 12 200953_s_at CCND2 -3.41 5.36E-11 4.68E-08 -1.91 -11.12 12p13 13 217716_s_at SEC61A1 -2.20 7.55E-13 2.14E-09 -1.72 -10.96 3q21.3 14 208852_s_at CANX -2.75 3.10E-12 6.16E-09 -1.70 -10.72 5q35 15 203948_s_at MPO -3.32 1.03E-12 2.56E-09 -1.66 -10.64 17q23.1 16 210788_s_at retSDR4 -2.44 1.63E-11 1.80E-08 -1.70 -10.51 14q22.3 17 AFFX- ACTB -2.29 1.37E-12 3.03E-09 -1.60 -10.33 7p15-p12 HSAC07/X00351 _M_at - HG-U133B 18 217225_x_at LOC283820 -2.14 4.38E-12 7.25E-09 -1.63 -10.32 16p13.13 19 214651_s_at HOXA9 165.30 1.46E-09 4.78E-07 2.15 10.16 7p15-p14 20 204150_at STAB1 -7.16 9.18E-10 3.88E-07 -1.80 -10.10 3p21.31 21 228760_at 6.67 8.42E-11 6.19E-08 1.65 10.00 22 213587_s_at LOC155066 5.20 9.89E-10 3.89E-07 1.83 9.97 7q36.1 23 229168_at DKFZp434K0621 -4.14 9.82E-10 3.89E-07 -1.73 -9.87 5q35.3 24 213106_at 4.55 5.67E-11 4.69E-08 1.60 9.86 25 205771_s_at AKAP7 12.68 1.65E-09 5.13E-07 1.85 9.83 6q23 26 213150_at HOXA10 31.06 2.47E-09 6.53E-07 1.96 9.81 7p15-p14 27 205382_s_at DF -2.68 3.99E-12 7.20E-09 -1.51 -9.80 19p13.3 28 AFFX- ACTB -2.16 5.86E-12 8.94E-09 -1.49 -9.62 7p15-p12 HSAC07/X00351 _M_at - HG-U133A 29 205663_at PCBP3 -3.00 2.34E-10 1.36E-07 -1.57 -9.57 21q22.3 30 211934_x__at G2AN -3.22 1.80E-10 1.19E-07 -1.56 -9.57 11q12.2 31 209215_at TETRAN -2.79 4.57E-11 4.32E-08 -1.51 -9.53 4p16.3 32 241383 at -3.94 5.71 E-09 1.27E-06 -1.80 -9.53 16 50 Tables 2 and 3 33 200951_s_at CCND2 -4.27 9.07E-10 3.88E-07 -1.62 -9.53 12p13 34 201596_x_at KRT18 -6.62 5.70E-10 2.83E-07 -1.59 -9.49 12q13 35 204425_at ARHGAP4 14.20 3.21 E-09 8.18E-07 1.79 9.49 Xq28 36 201OO4_at SSR4 -2.23 2.24E-10 1.36E-07 -1.54 -9.45 Xq28 37 226885_at 3.33 5.69E-10 2.83E-07 1.58 9.42 38 238365_s_at -3.90 7.10E-10 3.36E-07 -1.58 -9.41 39 211709_s_at SCGF -3.62 2.22E-11 2.32E-08 -1.46 -9.37 19q13.3 40 200047 s at - HG- YY1 1.88 1.47E-11 1.80E-08 1.44 9.33 14q U133A 41 208675_s_at DDOST -2.23 1.48E-11 1.80E-08 -1.44 -9.33 1p36.1 42 228046_at LOC 152485 4.74 3.68E-09 9.03E-07 1.71 9.29 4q31.1 43 200640_at YWHAZ -1.82 2.97E-11 2.94E-08 -1.44 -9.26 8q23.1 44 209344_at TPM4 -8.94 1.35E-08 2.57E-06 -1.80 -9.20 19p13.1 45 208689_s_at RPN2 -1.93 5.43E-11 4.68E-08 -1.43 -9.15 20q12- q13.1 46 227353_at EVER2 3.54 4.02E-10 2.22E-07 1.45 8.99 17q25.3 47 227326_at -3.53 2.12E-09 5.95E-07 -1.51 -8.98 48 209021_x_at KIAA0652 -3.43 2.28E-10 1.36E-07 -1.42 -8,96 11p11.12 49 229564_at dJ222E13.1 4.36 9.98E-10 3.89E-07 1.48 8.95 22q13 50 219837 s at C17 -11.98 2.18E-08 3.38E-06 -1.70 -8.85 4p16-p15
3.8 PTD versus t(821)
# affy id HUGO name fc P q stn t Map Location 1 213147_at HOXA10 12.09 1.79E-11 4.68E-07 2.20 12.11 7p15-p14 2 206847_s_at HOXA7 5.90 3.18E-11 4.68E-07 2.10 11.64 7p15-p14 3 235753_at 8.83 1.07E-10 1.04E-06 2.19 11.46 4 213908_at 7.63 5.10E-10 3.75E-06 1.86 10.22 5 214651_s_at HOXA9 141.82 1.49E-09 8.74E-06 2.13 10.15 7p15-p14 6 213150_at HOXA10 37.05 2.25E-09 1.10E-05 1.98 9.86 7p15-p14 7 201281_at ADRM1 -2.10 4.47E-09 1.88E-05 -1.54 -8.94 20q13.33 8 217963_s_at NGFRAP1 19.39 1.15E-08 2.60E-05 1.68 8.83 Xq22.1 9 206940_s_at POU4F1 -17.73 1.02E-07 1.20E-04 -1.77 -8.59 13q21.1- q22 10 211341_at POU4F1 -28.93 1.74E-07 1.74E-04 -1.75 -8.33 13q21.1- q22 11 228827_at -79.09 2.50E-07 2.04E-04 -1.90 -8.22 12 2099O5_at HOXA9 364.38 1.07E-07 1.21 E-04 1.67 7.87 7p15-p14 13 211728_s_at HYAL3 -3.88 8.31 E-08 1.02E-04 -1.35 -7.73 3p21.3 14 2056O0_x_at HOXB5 2.98 3.24E-08 5.02E-05 1.32 7.71 17q21.3 15 243806_at 4.67 6.31 E-08 8.43E-05 1.36 7.67 16 205529_s_at CBFA2T1 -12.57 6.09E-07 3.44E-04 -1.65 -7.65 8q22 17217520_x_at LOC283683 5.38 1.61 E-07 1.69E-04 1.54 7.64 15q11.2 18 226206_at FLJ32205 2.71 2.98E-08 4.87E-05 1.27 7.57 7p22.3 19 243010_at MSI2 3.10 8.08E-08 1.02E-04 1.33 7.54 17q23.1 20 AFFX- ACTB -1.94 9.85E-09 2.60E-05 -1.19 -7.46 7p15-p12 HSAC07/X00351 _M_at - HG-U133B 21 AFFX- ACTB -1.94 7.31 E-09 2.39E-05 -1.18 -7.45 7p15-p12 51 Tables 2 and 3 HSAC07/X00351_M_at - HG-U133A 22 210150_s_at LAMA5 -4.43 4.65E-07 2.97E-04 -1.41 -7.42 20q13.2- q13.3 23 AFFX- ACTB -1.28 1.15E-08 2.60E-05 -1.19 -7.41 7p15-p12 HSAC07/X00351_3_at - HG-U133A 24 218453_s_at C6orf35 1.62 7.91 E-09 2.39E-05 1.17 7.39 6q25.3 25 227853_at 2.48 8.14E-09 2.39E-05 1.16 7.36 26 224998_at CKLFSF4 2.27 1.85E-08 3.39E-05 1.18 7.34 16q21 27 219598 s at PTD013 1.80 1.31 E-08 2.76E-05 1.17 7.34 6q13- q22.33 28 205453_at H0XB2 18.65 2.99E-07 2.20E-04 1.47 7.34 17q21- q22 29 207839_s_at LOC51754 3.80 4.98E-08 7.32E-05 1.22 7.31 9p13.1 30 201288_at ARHGDIB -1.50 1.72E-08 3.37E-05 -1.16 -7.25 12p12.3 31 235521_at H0XA3 11.11 4.78E-07 2.99E-04 1.42 7.11 7p15-p14 32 205601_s_at HOXB5 3.02 2.30E-07 1.99E-04 1.25 7.09 17q21.3 33 210633_x_at KRT10 2.05 2.61 E-08 4.51 E-05 1.10 6.98 17q21- q23 34 233955_x_at HSPC195 3.02 2.47E-07 2.04E-04 1.21 6.97 5q31.3 35 228058_at LOG 124220 -2.78 3.08E-07 2.21 E-04 -1.17 -6.88 16p13.3 36 202315_s_at BCR -1.95 1.88E-07 1.74E-04 -1.14 -6.87 22q11.23 37 220558_x_at PHEMX 2.09 5.85E-08 8.19E-05 1.09 6.82 11p15.5 38 205528_s_at CBFA2T1 -33.41 2.96E-06 8.37E-04 -1.54 -6.82 8q22 39 205366_s_at HOXB6 35.11 1.11 E-06 5.01 E-04 1.40 6.75 17q21.3 40 218236_s_at PRKCN 3.85 1.59E-07 1.69E-04 1.10 6.74 2p21 41 233467_s_at PHEMX 2.23 1.90E-07 1.74E-04 1.09 6.68 11p15.5 42 239707_at FLJ25217 -4.23 1.60E-06 6.01 E-04 -1.21 -6.64 17p11.2 43 226235_at MGC17515 2.37 2.92E-07 2.20E-04 1.07 6.52 18p11.23 44 208146_s_at CPVL 11.95 1.58E-06 6.01 E-04 1.24 6.50 7p15-p14 45 228359_at KIAA1959 -2.35 8.71 E-07 4.44E-04 -1.10 -6.46 11q24.1 46 228345_at 2.77 2.88E-07 2.20E-04 1.05 6.46 47 202732_at PKIG 2.08 2.72E-07 2.16E-04 1.04 6.45 20q12- q13.1 48 232424_at PRDM16 9.40 1.72E-06 6.33E-04 1.21 6.44 1p36.23- p33 49 225765_at KPNB2 1.97 2.15E-07 1.92E-04 1.02 6.40 5q13.1 50 203859 s at PALM -3.60 2.99E-06 8.37E-04 -1.18 -6.40 19p13.3
3.9 PTD versus tMLL
# affy id HUGO name fc p q i stn t Map Location 1 228083_at CACNA2D4 -12.12 1.08E-09 1.24E-05 -1.44 -8.75 12p13.33 2 208116_s_at MAN1A1 3.86 1.69E-09 1.29E-05 1.37 8.74 6q22 3 214789_x_at SRP46 2.22 3.48E-11 8.01 E-07 1.20 8.56 11q22 4 200829_x_at ZNF207 1.65 8.45E-09 2.56E-05 1.18 7.80 17q11.2 5 201152_s_at MBNL1 -1.87 3.73E-09 1.71 E-05 -1.09 -7.51 3q25 6 205601_s_at HOXB5 3.26 1.43E-07 1.00E-04 1.32 7.48 17q21.3 7 220306_at FLJ20202 3.78 6.16E-08 8.34E-05 1.17 7.36 1p11.1 52 Tables 2 and 3 8218376_ _s_at MICAL -4.47 1.93E-08 3.70E-05 -1.10 -7.28 6q21 9226580_ .at BRMS1 1.96 8.23E-09 2.56E-05 1.04 7.26 14q13.1
10201105. .at LGALS1 -3.24 3.37E-09 1.71 E-05 -1.01 -7.19 22q13.1
11201151. _s_at MBNL1 -2.33 2.47E-08 4.07E-05 -1.07 -7.13 3q25
12205453. .at H0XB2 11.71 4.70E-07 1.86E-04 1.28 7.02 17q21- q22
13219360. _s_at TRPM4 -78.99 1.34E-07 1.00E-04 -1.29 -6.99 19q13.33
14228334. _x_at KIAA1712 1.86 8.90E-09 2.56E-05 0.98 6.98 4q34
15204082. .at PBX3 -3.01 2.43E-08 4.07E-05 -1.02 -6.98 9q33-q34
16218453. s_at C6orf35 1.56 1.55E-08 3.58E-05 0.99 6.94 6q25.3
17213159. .at PCNX -2.47 1.15E-08 2.93E-05 -0.96 -6.87 14q24.1
18227798.at 6.82 3.38E-07 1.68E-04 1.09 6.77
19201754. .at C0X6C -1.55 1.93E-08 3.70E-05 -0.95 -6.75 8q22-q23
20232424. .at PRDM16 13.67 1.05E-06 2.79E-04 1.32 6.75 1p36.23- p33
21201738. .at GC20 1.56 9.92E-08 9.93E-05 0.99 6.68 3p21.33
22205366. _s_at H0XB6 25.13 1.29E-06 3.02E-04 1.31 6.65 17q21.3
23225974. .at DKFZp762C1112 4.46 1.49E-07 1.00E-04 0.99 6.63 8q21.3
24232919. .at 2.17 1.07E-07 9.93E-05 0.96 6.56
25213737. _x_at -1.86 4.37E-08 6.28E-05 -0.93 -6.56
26200742. _s_at CLN2 -1.91 3.55E-08 5.45E-05 -0.92 -6.55 11p15
27221823. .at LOC90355 2.34 2.97E-07 1.68E-04 1.00 6.54 5q21.1
28212174. .at AK2 -2.78 1.05E-07 9.93E-05 -0.96 -6.54 1p34
29209605. .at TST -3.51 8.04E-08 9.93E-05 -0.95 -6.54 22q13.1
30226278. .at DKFZp313A2432 2.51 1.10E-07 9.93E-05 0.95 6.53 11p14.2
31230667. -at 1.53 1.38E-07 1.00E-04 0.95 6.51
32222761. -at BIVM 2.73 3.08E-07 1.68E-04 0.99 6.51 13q32- q33.1
33225464. -at C14orf31 2.61 9.25E-08 9.93E-05 0.93 6.48 14q21.3
34202318. _s_at SUSP1 -2.08 1.15E-07 9.93E-05 -0.94 -6.45 6q13- q14.3
35232038. -at 2.51 3, 16E-07 1.68E-04 0.97 6.44
36228652. .at FLJ38288 1.81 1.17E-07 9.93E-05 0.93 6.44 19q13.43
37205600. _x_at H0XB5 2.14 1.49E-06 3.20E-04 1.13 6.43 17q21.3
38229143. _at CN0T3 1.91 1.52E-07 1.00E-04 0.93 6.42 19q13.4
39221760. -.at MAN1A1 4.55 1.11 E-06 2.81 E-04 1.07 6.42 6q22
40213258. .at 8.86 1.87E-06 3.48E-04 1.15 6.39
41213152. _s_at SRP46 2.59 3.91 E-07 1.71 E-04 0.96 6.37 11q22
42227400. -at NFIX 4.61 6.59E-07 2.14E-04 1.00 6.37 19p13.3
43230006. _s_at DKFZp313A2432 2.41 2.27E-07 1.41 E-04 0.92 6.33 11p14.2
44221235. _s_at 1.99 9.97E-07 2.73E-04 1.01 6.32
45218718. -.at PDGFC 3.45 8.73E-08 9.93E-05 0.88 6.29 4q32
46216941. s_at TAF1 B -1.80 8.73E-08 9.93E-05 -0.88 -6.29 2p25
47228974. .at 3.57 1.56E-06 3.22E-04 1.05 6.28
48228760. .at 2.74 1.32E-07 1.00E-04 0.89 6.28
49244103. .at 2.45 8.00E-07 2.36E-04 0.97 6.26
50226517 at BCAT1 6.88 2, 15E-06 3.82E-04 1.08 6.24 12pter- q12 Tables 2 and 3
3.10 inv3 versus t(15;17)
# affy id HUGO name fc P q stn t Map Location 1 212953_x_at CALR -5.95 2.17E-14 5.07E-11 -3.69 -18.88 19p13.3- p13.2 2 205382_s_at DF -12.24 2.37E-15 7.12E-12 -3.43 -18.68 19p13.3 3 203948_s_at MPO -9.29 4.98E-19 1.05E-14 -3.14 -18.57 17q23.1 4 203949_at MPO -6.22 1.52E-17 1.60E-13 -3.05 -17.82 17q23.1 5 200654_at P4HB -3.78 4.67E-17 3.27E-13 -2.71 -16.03 17q25 6 214450_at CTSW -8.62 1.58E-13 2.89E-10 -2.90 -15.67 11q13.1 7 231736_x_at MGST1 -6.90 6.57E-16 2.30E-12 -2.57 -15.09 12p12.3- p12.1 8 224918_x_at MGST1 -6.02 2.58E-16 1.15E-12 -2.54 -15.02 12p12.3- p12.1 9 206871 _at ELA2 -6.28 2.73E-16 1.15E-12 -2.54 -15.00 19p13.3 10 214575_s_at AZU1 -12.19 2.49E-13 3.73E-10 -2.58 -14.34 19p13.3 11 205624_at CPA3 -21.54 5.79E-12 5.79E-09 -2.85 -14.33 3q21-q25 12 208689_s_at RPN2 -2.77 3.65E-15 9.58E-12 -2.43 -14.27 20q12- q13.1 13 238022_at -8.14 1.08E-12 1.33E-09 -2.28 -12.89 14 38487_at STAB1 -5.21 5.94E-13 8.31 E-10 -2.23 -12.76 3p21.31 15 221004_s_at ITM2C -4.36 8.93E-14 1.88E-10 -2.12 -12.49 2q37 16 217716_s_at SEC61A1 -2.51 1.65E-13 2.89E-10 -2.09 -12.25 3q21.3 17 221739_at IL27w -2.24 2.31 E-13 3.73E-10 -2.06 -12.11 19p13.3 18 233072_at KIAA1857 -10.04 1.05E-10 5.14E-08 -2.37 -12.06 9q34 19 208852_s_at CANX -2.94 3.24E-12 3.78E-09 -2.07 -11.86 5q35 20 220798_x_at FLJ11535 -5.26 7.78E-12 6.81 E-09 -2.05 -11.62 19p13.3 21 217225_x_at LOC283820 -2.41 9.52E-13 1.25E-09 -1.94 -11.43 16p13.13 22 208730_x_at RAB2 2.53 8.63E-10 3.12E-07 2.18 11.42 8q12.1 23 203675_at NUCB2 -3.92 6.96E-12 6.65E-09 -2.00 -11.42 11p15.1- p14 24 201004_at SSR4 -2.77 1.64E-11 1.15E-08 -2.00 -11.33 Xq28 25 210788_s_at retSDR4 -2.65 7.69E-12 6.81 E-09 -1.95 -11.22 14q22.3 26 202759_s_at AKAP2 -4.78 2.58E-11 1.69E-08 -1.98 -11.15 9q31-q33 27 209619_at CD74 4.57 1.47E-11 1.14E-08 1.92 11.07 5q32 28 214315_x_at CALR -3.14 2.25E-11 1.52E-08 -1.93 -11.00 19p13.3- p13.2 29 229168_at DKFZp434K0621 -5.62 4.18E-10 1.72E-07 -2.12 -10.99 5q35.3 30 211990_at HLA-DPA1 12.02 1.70E-08 3.31 E-06 2.38 10.92 6p21.3 31 214797_s_at PCTK3 6.22 2.95E-09 8.48E-07 2.12 10.91 1q31-q32 32 211709_s_at SCGF -5.08 3.77E-12 3.96E-09 -1.80 -10.65 19q13.3 33 200068 s at - HG- CANX -1.76 3.59E-12 3.96E-09 -1.79 -10.61 5q35 U133A 34 206914_at CRTAM 6.82 3.01 E-09 8.54E-07 1.99 10.50 11 q22- q23 35 204897_at PTGER4 5.48 3.25E-10 1.37E-07 1.87 10.44 5p13.1 36 221253_s_at MGC3178 -3.45 5.95E-11 3.62E-08 -1.81 -10.36 6p24.3 37 225010_at D10S170 2.56 2.69E-11 1.71 E-08 1.77 10.33 10q21 38 210140_at CST7 -8.79 1.17E-09 4.09E-07 -1.98 -10.32 20p11.21 39 226905_at -1.96 8.40E-11 4.20E-08 -1.78 -10.24 40 200652_at SSR2 -1.91 1.02E-11 8.61 E-09 -1.73 -10.22 1q21-q23 54 Tables 2 and 3 41 33323_r_at SFN 1.93 1.07E-11 8.68E-09 1.73 10.21 1p35.3 42 227353_at EVER2 5.28 1.34E-08 2.75E-06 2.02 10.17 17q25.3 43 224839_s_at GPT2 -6.13 8.34E-11 4.20E-08 -1.77 -10.15 16q12.1 44 200068 s at - HG- CANX -1.67 1.62E-11 1.15E-08 -1.72 -10.14 5q35 U133B 45209215_at TETRAN -3.38 1.52E-11 1.14E-08 -1.72 -10.14 4p16.3 46 205614_x_at MST1 -8.62 3.49E-09 9.53E-07 -2.00 -9.99 3p21 47 241383_at -4.56 2.13E-09 6.47E-07 -1.87 -9.85 48 214317_x_at RPS9 2.30 1.38E-09 4.55E-07 1.77 9.82 19q13.4 49 202487_s_at H2AV -2.25 6.02E-11 3.62E-08 -1.64 -9.66 7p13 50 204661_at CDW52 22.88 1.06E-07 1.35E-05 2.16 9.63 1p36
3.11 inv3 versus t(821)
# affy id HUGO name fc P q stn t Map Location 1 203949_at MPO -5.65 7.52E-13 1.81 E-08 -2.11 -12.02 17q23.1 2 211084_x_at PRKCN 5.87 3.47E-10 2.79E-06 1.89 10.40 2p21 3 233955_x_at HSPC195 5.22 3.15E-08 8.44E-05 2.17 10.18 5q31.3 4 225010_at D10S170 2.88 2.98E-11 3.60E-07 1.75 10.06 10q21 5 203948_s_at MPO -6.72 6.92E-10 4.17E-06 -1.71 -9.50 17q23.1 6 201281_at ADRM1 -2.23 1.63E-09 7.87E-06 -1.63 -9.09 20q13.33 7 217963_s_at NGFRAP1 29.06 4.70E-07 3.72E-04 2.04 8.66 Xq22.1 8 217226_s_at BA108L7.2 3.73 5.93E-08 1.43E-04 1.66 8.63 10q24.31 9 219478_at WFDC1 -12.65 9.84E-08 1.98E-04 -1.72 -8.45 16q24.3 10 224516_s_at HSPC195 5.79 5.70E-07 3.72E-04 1.91 8.42 5q31.3 11 231180_at -2.39 2.87E-09 1.15E-05 -1.47 -8.39 12 228827_at -99.36 2.41 E-07 3.23E-04 -1.91 -8.24 13 222996_s_at HSPC195 4.30 1.07E-06 5.36E-04 1.78 7.97 5q31.3 14 212423_at FLJ90798 4.16 7.34E-08 1.61 E-04 1.47 7.96 10q22.3 15 230259_at -1.94 2.68E-08 8.08E-05 -1.41 -7.87 16 220974_x_at BA108L7.2 5.01 4.47E-07 3.72E-04 1.57 7.86 10q24.31 17 230659_at KIAA0212 -2.16 1.23E-07 2.29E-04 -1.47 -7.79 3p26.1 18 202759_s_at AKAP2 -5.05 2.41 E-07 3.23E-04 -1.52 -7.74 9q31-q33 19 205529_s_at CBFA2T1 -14.01 5.55E-07 3.72E-04 -1.74 -7.73 8q22 20 213716_s_at SECTM1 4.82 2.88E-07 3.30E-04 1.42 7.55 17q25 21 206478_at KIAA0125 23.37 2.67E-06 8.04E-04 1.89 7.54 14q32.33 22 219165_at PDLIM2 3.74 6.52E-07 4.03E-04 1.46 7.47 8p21.2 23 211709_s_at SCGF -3.56 2.43E-08 8.08E-05 -1.29 -7.41 19q13.3 24 212895_s_at ABR 3.07 3.51 E-07 3.53E-04 1.38 7.36 17p13.3 25 203820_s_at KOC1 4.07 2.40E-06 7.71 E-04 1.56 7.29 7p11 26 206295_at IL18 3.55 2.33E-06 7.62E-04 1.53 7.25 11q22.2- q22.3 27 210150_s_at LAMA5 -4.29 4.79E-07 3.72E-04 -1.38 -7.22 20q13.2- q13.3 28 201243_s_at ATP1 B1 5.05 2.16E-06 7.35E-04 1.49 7.20 1q22-q25 29 202006_at PTPN12 2.72 7.49E-07 4.41 E-04 1.37 7.18 7q11.23 30 202887 s at RTP801 4.33 1.63E-06 6.56E-04 1.43 7.14 10pter- q26.12 55 Tables 2 and 3 31 207839_s_at LOC51754 3.10 2.62E-07 3.30E-04 1.28 7.08 9p13.1 32 201938_at CDK2AP1 2.04 1.33E-07 2.29E-04 1.25 7.07 12q24.31 33 214042_s_at RPL22 1.48 8.29E-07 4.76E-04 1.33 7.04 1p36.3- p36.2 34 226865_at 8.77 5.57E-06 1.18E-03 1.65 7.01 35 222955_s_at HT011 -2.21 5.13E-07 3.72E-04 -1.31 -7.01 Xq26.1 36 242621_at FLJ32468 -1.60 3.47E-07 3.53E-04 -1.28 -7.00 7q22.1 37 223534_s_at RPS6KL1 -2.19 3.25E-07 3.53E-04 -1.28 -7.00 14q24.2 38 215051_x_at AIF1 2.61 2.78E-07 3.30E-04 1.26 6.99 6p21.3 39 231334_at -3.75 8.70E-07 4.86E-04 -1.35 -6.98 40 2139O8_at 4.04 2.34E-06 7.62E-04 1.38 6.94 41 204494_s_at DKFZP434H132 5.00 5.81 E-06 1.22E-03 1.57 6.92 15q22.33 42 212953_x_at CALR -2.43 1.33E-06 5.73E-04 -1.37 -6.92 19p13.3- p13.2 43 228058_at LOC124220 -2.64 5.60E-07 3.72E-04 -1.28 -6.91 16p13.3 44 227620_at 3.61 5.09E-07 3.72E-04 1.25 6.87 45 221458_at HTR1F 2.61 1.74E-06 6.76E-04 1.32 6.86 3p12 46 221773_at 3.67 1.02E-06 5.26E-04 1.28 6.85 47 210613_s_at SYNGR1 -2.93 1.84E-07 2.96E-04 -1.20 -6.83 22q13.1 48 214807_at 2.96 2.54E-06 8.04E-04 1.35 6.83 49 229406_at -11.96 2.16E-06 7.35E-04 -1.40 -6.81 50 205528_s_at CBFA2T1 -30.57 3.06E-06 8.69E-04 -1.54 -6.79 8q22
3.12 inv3 versus tMLL
# affy id HUGO name fc P q stn t Map Location 1 204082_at PBX3 -8.13 5.43E-11 4.44E-07 -1.62 -9.85 9q33-q34 2 233955_x_at HSPC195 5.24 8.76E-09 7.67E-06 1.68 9.58 5q31.3 3 226789_at -3.29 1.13E-11 2.40E-07 -1.47 -9.56 4 214651_s_at HOXA9 -4.39 1.96E-11 2.40E-07 -1.37 -9.06 7p15-p14 5 225344_at ERAP140 4.30 2.49E-07 5.42E-05 1.75 8.68 6q22.33 6 236398_s_at -6.51 6.25E-10 2.19E-06 -1.32 -8.42 7235753_at -4.84 4.98E-10 2.03E-06 -1.30 -8.41 8 2100O6_at DKFZP5640243 -2.26 4.92E-10 2.03E-06 -1.29 -8.36 3p21.1 9 224516_s_at HSPC195 6.41 2.69E-07 5.68E-05 1.59 8.32 5q31.3 10 222982_x_at SLC38A2 1.92 1.09E-09 2.44E-06 1.29 8.32 12q 11 235199_at 3.81 2.04E-07 4.75E-05 1.54 8.30 12 213893_x_at PMS2L5 -2.33 3.45E-10 2.03E-06 -1.25 -8.22 7q11-q22 13 214643_x_at BIN1 4.75 2.15E-07 4.88E-05 1.51 8.20 2q14 14 203733_at MYLE -2.90 7.44E-10 2.28E-06 -1.25 -8.16 16p13.2 15 2099O5_at HOXA9 -6.88 1.31 E-09 2.44E-06 -1.27 -8.14 7p15-p14 16 212782_x_at POLR2J -2.47 1.59E-09 2.44E-06 -1.24 -8.04 7q11.2 17 228083_at CACNA2D4 -8.54 2.08E-09 2.83E-06 -1.26 -8.04 12p13.33 18 202961_s_at ATP5J2 -2.29 1.53E-09 2.44E-06 -1.23 -8.03 7q22.1 19 225386_s_at LOC92906 -6.33 1.09E-09 2.44E-06 -1.22 -7.98 2p22.2 20 212318_at TRN-SR -2.60 1.30E-09 2.44E-06 -1.21 -7.92 7q32.2 21 211978_x_at PPIA -1.66 4.91 E-09 5.47E-06 -1.23 -7.89 7p13- p11.2 22 56 Tables 2 and 3 22 222996_s_at HSPC195 4.55 5.97E-07 9.52E-05 1.52 7.89 5q31.3 23 223207_x_at PHP14 -1.83 1.21 E-09 2.44E-06 -1.17 -7.76 9q34.3 24 208116_s_at MA 1A1 4.91 8.97E-07 1.19E-04 1.53 7.75 6q22 25 223703_at CDA017 -3.77 6.76E-09 6.56E-06 -1.24 -7.75 10q23.1 26 211378_x_at PPIA -1.67 1.05E-08 8.33E-06 -1.21 -7.74 7p13- p11.2 27 200602_at APP 9.66 7.49E-07 1.08E-04 1.47 7.70 21q21.3 28 212174_at AK2 -3.81 4.84E-09 5.47E-06 -1.20 -7.70 1p34 29 214430_at GLA -2.12 1.56E-09 2.44E-06 -1.16 -7.68 Xq22 30 202053_s_at ALDH3A2 -2.84 6.95E-09 6.56E-06 -1.21 -7.66 17p11.2 31 202054_s_at ALDH3A2 -4.35 1.85E-09 2.67E-06 -1.16 -7.65 17p11.2 32 214453_s_at IFI44 5.44 1.39E-06 1.62E-04 1.56 7.63 1p31.1 33 201293_x_at PPIA -1.61 1.25E-08 9.18E-06 -1.19 -7.61 7p13- p11.2 34 209836_x_at MGC5178 -2.07 2.21 E-09 2.85E-06 -1.15 -7.61 16p12.1 35 208967_s_at AK2 -3.93 1.89E-08 1.13E-05 -1.23 -7.52 1p34 36 230051_at 4.17 4.75E-07 8.15E-05 1.31 7.43 37 202605_at GUSB -3.22 6.78E-09 6.56E-06 -1.13 -7.38 7q21.11 38 225389_at BTBD6 -2.28 4.68E-09 5.47E-06 -1.11 -7.35 14q32 39 219551_at TRAITS -3.19 1.15E-08 8.82E-06 -1.14 -7.34 3q13.33 40 201829_at NET1 3.64 2.56E-06 2.40E-04 1.53 7.32 10p15 41 206478_at KIAA0125 15.02 3.35E-06 2.89E-04 1.66 7.32 14q32.33 42 201186_at LRPAP1 -3.24 1.57E-08 1.01 E-05 -1.14 -7.31 4p16.3 43 219126_at XAP135 -1.82 5.33E-09 5.68E-06 -1.10 -7.31 6q27 44 223328_at MGC3195 -2.10 9.36E-09 7.92E-06 -1.11 -7.30 7q22.1 45 211765_x_at PPIA -1.60 4.19E-08 1.88E-05 -1.15 -7.30 7p13- p11.2 46 205514_at FLJ11191 4.23 1.61 E-06 1.78E-04 1.40 7.29 19q13.41 47 215667_x_at PMS2L5 -1.92 7.42E-09 6.74E-06 -1.10 -7.27 7q11-q22 48 212661_x_at -1.59 4.26E-08 1.88E-05 -1.13 -7.21 49 228652_at FLJ38288 2.29 6.02E-07 9.52E-05 1.26 7.20 19q13.43 50 213908 at -3.92 3.73E-08 1.83E-05 -1.16 -7.20
3.13 t(15;17) versus t(821)
# affy id HUGO name fc P q stn Map Location 1 214450_at CTSW 27.45 1.67E-13 5.02E-09 3.57 17.69 11q13.1 2 38487_at STAB1 19.09 4.71 E-13 7.07E-09 3.25 16.45 3p21.31 3 209732_at CLECSF2 -30.85 1.79E-11 4.88E-08 -3.32 ■15.30 12p13- p12 4 211990_at HLA-DPA1 -11.19 1.56E-11 4.67E-08 -2.71 •14.09 6p21.3 5 224839_s_at GPT2 12.86 6.29E-11 1.35E-07 2.35 12.29 16q12.1 6 212509_s_at 9.95 9.86E-11 1.96E-07 2.36 12.15 7 226878_at -5.69 4.61 E-10 5.32E-07 -2.25 ■11.62 8 204150_at STAB1 20.67 3.59E-10 4.49E-07 2.35 11.56 3p21.31 9 201596_x_at KRT18 20.76 3.10E-10 4.05E-07 2.28 11.50 12q13 10 205349_at GNA15 3.49 3.36E-11 8.40E-08 1.98 11.31 19p13.3 11 205663 at PCBP3 4.59 8.09E-12 3.47E-08 1.92 11.31 21q22.3 57 Tables 2 and 3 12221004. _s_at ITM2C 3.23 8.52E-13 8.53E-09 1.85 11.27 2q37 13212953. _x_at CALR 2.45 1.20E-12 8.99E-09 1.76 10.80 19p13.3- p13.2 14217478.s_at HLA-DMA -5.51 4.09E-10 4.91 E-07 -1.94 10.73 6p21.3 15227326.at 5.33 2.87E-10 3.99E-07 1.88 10.49 16228113.at STAT3 -5.22 9.54E-10 8.68E-07 -1.92 ■10.46 17q21 17217716. _s_at SEC61A1 2.04 7.06E-12 3.47E-08 1.71 10.39 3q21.3 18208826. _x_at HINT1 1.40 4.69E-12 2.81 E-08 1.68 10.32 5q31.2 19200986.at SERPING1 9.53 1.51 E-09 1.26E-06 1.97 10.29 11q12- q13.1 20201137. _s_at HLA-DPB1 -13.90 1.17E-08 6.03E-06 -2.10 •10.00 6p21.3 21208689. _s_at RPN2 1.78 1.23E-11 4.60E-08 1.60 9.83 20q12- q13.1 22204316. .at RGS10 -2.46 9.39E-10 8.68E-07 -1.71 -9.76 10q25 23209619. .at CD74 -4.69 2.02E-10 3.19E-07 -1.65 -9.75 5q32 24204670. _x_at HLA-DRB5 -5.88 5.55E-10 5.74E-07 -1.68 -9.73 6p21.3 25201522. _x_at SNRPN -3.71 1.47E-11 4.67E-08 -1.58 -9.71 15q12 26211991. _s_at HLA-DPA1 -17.64 1.79E-08 8.26E-06 -2.00 -9.66 6p21.3 27205614. _x_at MST1 7.48 3.65E-09 2.28E-06 1.82 9.65 3p21 28209021. _x_at KIAA0652 4.23 5.35E-11 1.23E-07 1.59 9.63 11p11.12 29200953. _s_at CCND2 2.65 5.21 E-10 5.74E-07 1.64 9.52 12p13 30209312. _x_at HLA-DRB1 -7.00 4.59E-09 2.76E-06 -1.73 -9.47 6p21.3 31208852. _s_at CANX 2.27 1.04E-10 1.96E-07 1.56 9.42 5q35 32201425. .at ALDH2 5.10 1.1 OE-09 9.46E-07 1.60 9.25 12q24.2 33201136. .at PLP2 2.70 2.93E-10 3.99E-07 1.54 9.23 Xp11.23 34201952. _at ALCAM 4.55 2.64E-09 1.80E-06 1.63 9.21 3q13.1 35218795. _at ACP6 -2.74 3.46E-09 2.21 E-06 -1.61 -9.16 1q21 36208306. _x_at HLA-DRB4 -7.29 1.08E-08 5.68E-06 -1.69 -9.14 6p21.3 37206940. _s_at P0U4F1 -45.95 7.19E-08 1.96E-05 -2.09 -8.99 13q21.1- q22 38223321. _s_at FGFRL1 3.71 4. 93E-09 2.90E-06 1.59 8.94 4p16 39201923. .at PRDX4 -5.97 1.90E-08 8.63E-06 -1.67 -8.94 Xp22.13 40215193. x_at HLA-DRB1 -7.01 5.77E-09 3.27E-06 -1.57 -8.92 6p21.3 41207721. _x_at HINT1 1.51 1.53E-10 2.70E-07 1.45 8.89 5q31.2 42238022. _at 3.92 1.64E-10 2.74E-07 1.43 8.81 43227353. -.at EVER2 -3.90 8.45E-09 4.61 E-06 -1.56 -8.81 17q25.3 44224451. _x_at ARHGAP9 -2.71 7, 08E-10 6.86E-07 -1.45 -8.77 12q14 45209344. -.at TPM4 6.76 2.31 E-08 9.73E-06 1.68 8.76 19p13.1 46211474. _s_at SERPINB6 -5.75 5.58E-08 1.75E-05 -1.73 -8.75 6p25 47201360. --at CST3 4.40 2.09E-09 1.53E-06 1.48 8.70 20p11.21 48201894. _s_at DCN 1.99 2.33E-10 3.49E-07 1.41 8.69 12q13.2 49202732. --at PKIG 2.65 2, 59E-09 1.80E-06 1.48 8.66 20q12- q13.1 50211341 at P0U4F1 1.24E-07 2.80E-05 -2.02 -8.65 13q21.1- 309.60 q22
3.14 t(15;17) versus tMLL
# affy id HUGO name fc stn t Map Location 58 Tables 2 and 3 1 221004_s_at ITM2C 10.63 1.44E-14 5.99E-11 2.85 16.93 2q37 2 38487_at STAB1 16.43 2.86E-13 5.50E-10 2.90 16.09 3p21.31 3 205624_at CPA3 36.17 5.95E-12 7.42E-09 3.00 14.74 3q21-q25 4 203948_s_at MPO 5.78 4.02E-19 1.00E-14 2.09 14.65 17q23.1 5 214651_s_at HOXA9 2.65E-14 9.43E-11 -2.61 -14.18 7p15-p14 236.49 6 212953_x_at CALR 3.23 1.31 E-14 5.99E-11 2.20 14.16 19p13.3- p13.2 7 214450_at CTSW 6.15 3.95E-14 1.23E-10 2.18 13.92 11q13.1 8 200953_s_at CCND2 6.53 2.29E-12 3.11 E-09 2.32 13.58 12p13 9 203949_at MPO 4.10 5.38E-16 4.47E-12 1.82 12.55 17q23.1
10 206871_at ELA2 4.27 1.94E-16 2.43E-12 1.79 12.53 19p13.3
11 238022_at 6.11 2.20E-12 3.11 E-09 1.97 12.29
12 233072_at KIAA1857 12.53 6.26E-11 3.19E-08 2.25 12.28 9q34
13 213147_at HOXA10 -23.65 1.43E-12 2.37E-09 -2.06 -11.90 7p15-p14
14 204150_at STAB1 20.35 3.43E-10 1.16E-07 2.25 11.53 3p21.31
15 209448_at HTATIP2 -10.10 2.27E-12 3.11 E-09 -1.86 -11.35 11p15.1
16 200951_s_at CCND2 7.49 1.92E-10 7.62E-08 1.99 11.24 12p13
17 210788_s_at retSDR4 2.54 1.20E-11 1.17E-08 1.77 11.17 14q22.3
18 201029_s_at CD99 2.20 1.43E-14 5.99E-11 1.57 11.01 Xp22.32
19 205663_at PCBP3 3.90 2.95E-11 2.05E-08 1.77 11.01 21q22.3
20 205349_at GNA15 4.35 9.31 E-13 1.66E-09 1.64 10.95 19p13.3
21 212509_s_at 6.22 1.29E-10 5.66E-08 1.84 10.93
22 206761_at TACTILE 29.33 1.19E-09 3.13E-07 2.28 10.90 3q13.13
23 200952_s_at CCND2 4.35 2.19E-10 8.42E-08 1.86 10.85 12p13
24 201596_x_at KRT18 10.40 5.91 E-10 1.85E-07 1.98 10.82 12q13
25 217848_s_at PP -3.83 1.98E-13 4.18E-10 -1.59 -10.82 10q11.1- q24
26 235753_at -16.42 1.82E-11 1.42E-08 -1.91 -10.74
27 206847_s_at HOXA7 -9.06 7.47E-12 8.47E-09 -1.72 -10.70 7p15-p14
28 201522_x_at SNRPN -4.71 6.74E-14 1.87E-10 -1.53 -10.64 15q12
29 225532_at LOC91768 5.85 7.26E-10 2.16E-07 1.93 10.63 18q11.1
30 205771_s_at AKAP7 -9.87 9.16E-12 9.94E-09 -1.70 -10.58 6q23
31 231736_x_at MGST1 2.94 2.01 E-13 4.18E-10 1.53 10.56 12p12.3- p12.1
32 213587_s_at LOC 155066 -7.85 2.11E-11 1.58E-08 -1.79 -10.53 7q36.1
33 213150_at HOXA10 -43.13 3.71 E-11 2.32E-08 -1.85 -10.41 7p15-p14
34 224918_x_at MGST1 2.74 1.26E-13 3.15E-10 1.48 10.35 12p12.3- p12.1
35 225386_s_at LOC92906 -36.93 5.16E-11 2.86E-08 -1.80 -10.23 2p22.2
36 209905_at HOXA9 6.49E-11 3.24E-08 -1.89 -10.18 7p15-p14 700.51
37 221253_s_at MGC3178 3.07 1.58E-10 6.70E-08 1.60 10.05 6p24.3
38 204082_at PBX3 -8.66 4.80E-11 2.73E-08 -1.63 -9.98 9q33-q34
39 218404_at SNX10 -6.62 3.43E-11 2.19E-08 -1.59 -9.95 7p15.2
40 225653_at 2.34 1.14E-09 3.02E-07 1.64 9.75
41 217716_s_at SEC61A1 1.98 7.04E-12 8.37E-09 1.43 9.71 3q21.3
42 219837_s_at C17 88.02 8.44E-09 1.29E-06 2.06 9.69 4p16-p15
43 202265_at BMI1 -4.05 2.73E-11 1.95E-08 -1.48 -9.68 10p11.23
44 212813_at JAM3 5.10 3.78E-09 7.19E-07 1.74 9.64 11q25
45 241383 at 4.10 4.07E-09 7.52E-07 1.74 9.61 59 Tables 2 and 3 46 210140_at CST7 6.56 1.35E-09 3.32E-07 1.59 9.54 20p11.21 47 202746_at ITM2A 18.72 8.03E-09 1.26E-06 1.84 9.53 Xq13.3- Xq21.2 48 225570_at SLC41A1 -3.47 1.50E-11 1.34E-08 -1.40 -9.47 1q32.1 49 211474_s_at SERPINB6 -4.69 7.52E-11 3.54E-08 -1.47 -9.45 6p25 50 208852_s_at CANX 2.24 7.24E-11 3.48E-08 1.43 9.44 5q35
3.15 t(821) versus tMLL
# affy id HUGO name fc P q stn t Map Location 1 214651_s_at HOXA9 2.68E-14 7.75E-10 -2.60 -14.17 7p15-p14 202.90 2 221581_s_at WBSCR5 -9.72 2.43E-13 3.51 E-09 -2.04 -12.41 7q11.23 3 213147_at HOXA10 -14.91 2.06E-12 1.19E-08 -1.91 -11.48 7p15-p14 4 201105_at LGALS1 -6.99 4.93E-13 4.74E-09 -1.67 -10.99 22q13.1 5 235753_at -14.41 2.17E-11 7.40E-08 -1.87 -10.63 6 206847_s_at HOXA7 -7.77 1.54E-11 6.34E-08 -1.77 -10.59 7p15-p14 7 213150_at HOXA10 -51.45 3.41 E-11 8.96E-08 -1.87 -10.45 7p15-p14 8 209905_at HOXA9 6.52E-11 1.52E-07 -1.89 -10.18 7p15-p14 608.56 9 227853_at -3.66 1.21 E-12 8.72E-09 -1.47 -9.96 10 210314_x_at TNFSF13 -4.46 2.53E-11 7.40E-08 -1.42 -9.38 17p13.1 11 213908_at -15.84 3.74E-10 5.69E-07 -1.63 -9.33 12 203949_at MPO 3.73 1.28E-11 6.15E-08 1.34 9.15 17q23.1 13 216417_x_at HOXB9 -3.57 2.56E-11 7.40E-08 -1.36 -9.13 17q21.3 14 228058_at LOC 124220 6.98 1.09E-09 9.83E-07 1.42 8.99 16p13.3 15 209500_x_at TNFSF13 -3.79 1.57E-10 3.03E-07 -1.39 -8.99 17p13.1 16 204082_at PBX3 -6.21 1.07E-10 2.21 E-07 -1.37 -8.98 9q33-q34 17 206940_s_at POU4F1 42.83 7.36E-08 2.10E-05 2.07 8.97 13q21.1- q22 18 225245_x_at H2AFJ -5.25 2.55E-10 4.61 E-07 -1.37 -8.84 12p12 19 228083_at CACNA2D4 -12.80 1.04E-09 9.71 E-07 -1.50 -8.84 12p13.33 20 211341_at POU4F1 239.48 1.25E-07 2.94E-05 2.01 8.64 13q21.1- q22 21 228365_at LOC144402 -7.69 1.63E-09 1.38E-06 -1.44 -8.60 12q11 22 202746_at ITM2A 7.78 2.76E-08 1.01 E-05 1.49 8.56 Xq13.3- Xq21.2 23 212459_x_at SUCLG2 -3.83 6.86E-11 1.52E-07 -1.25 -8.53 3p14.2 24 218404_at SNX10 -4.04 4.74E-10 6.85E-07 -1.31 -8.53 7p15.2 25 201944_at HEXB -3.46 1.87E-09 1.46E-06 -1.39 -8.47 5q13 26 223562_at PARVG -3.25 6.78E-10 8.16E-07 -1.30 -8.43 22q 13.2- q13 27 204202_at KIAA1023 -3.48 6.42E-10 8.06E-07 -1.28 -8.38 7p22.3 28 212423_at FLJ90798 -5.34 6.18E-10 8.06E-07 -1.28 -8.37 10q22.3 29 205639_at AOAH -5.26 7.95E-10 8.83E-07 -1.26 -8.27 7p14-p12 30 224301_x_at H2AFJ -4.35 8.84E-10 9.12E-07 -1.26 -8.26 12p12 31 228827_at 114.73 2.37E-07 4.10E-05 1.93 8.26 32 201850_at CAPG -8.11 4.48E-09 2.88E-06 -1.40 -8.24 2cen-q24 33 208890_s_at PLXNB2 -4.00 9.67E-10 9.31 E-07 -1.24 -8.17 22q13.33 34 221841 s at -4.00 3.65E-10 5.69E-07 -1.20 -8.13 60 Tables 2 and 3
35 214835_s_at SUCLG2 -4.02 7.31 E-10 8.45E-07 -1.21 -8.10 3p14.2
36 224415_s_at HINT2 -2.05 3.35E-10 5.69E-07 -1.18 -8.08 9p13.1
37 201281_at ADRM1 1.93 1.65E-08 7.22E-06 1.29 8.04 20q13.33
38 218217_at RISC -5.08 3.89E-09 2.69E-06 -1.28 -8.04 17q23.1
39 238756_at -4.18 2.31 E-09 1.76E-06 -1.24 -8.01
40 242931_at -3.58 1.78E-09 1.43E-06 -1.22 -7.99
41 204069_at MEIS1 -17.90 1.09E-08 5.54E-06 -1.42 -7.96 2p14-p13
42 241370_at -3.07 3.39E-09 2.45E-06 -1.24 -7.96
43 225386_s_at LOC92906 -6.56 9.62E-10 9.31 E-07 -1.17 -7.91 2p22.2
44 215772_x_at SUCLG2 -4.01 5.45E-10 7.49E-07 -1.15 -7.88 3p14.2
45 229002_at MGC20262 4.77 9.02E-08 2.39E-05 1.35 7.88 9q34.3
46 219478_at WFDC1 7.40 2.12E-07 3.90E-05 1.44 7.84 16q24.3
47 213737_x_at -1.99 8.41 E-10 9.00E-07 -1.14 -7.80
48 221760_at MAN1A1 12.13 4.59E-07 6.50E-05 1.62 7.78 6q22
49 219271_at GalNac-T10 6.98 2.26E-07 4.00E-05 1.41 7.76 2p23.1
50 231334 at 5.10 2.43E-07 4.16E-05 1.42 7.75

Claims

Claims
1. A method for distinguishing MLL-PTD-positive AML from other AML subtypes in a sample, the method comprising determining the expression level of markers selected from the markers identifiable by their Affymetrix Identification Numbers (affy id) as defined in Tables 1, 2, and/or 3, wherein a lower expression of at least one polynucleotide defined by any of the numbers 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, and/or 50 of Table 1 is indicative for the presence of PTD (MLL-PTD-positive AML with normal karyotype) when PTD is distinguished from AML_NK (MLL- PTD-negative AML with normal karyotype), and/or wherein a lower expression of at least one polynucleotide defined by any of the numbers 1, 2, 3, 4, 5, 6, 7, 8, 9, 11, 12, 14, 15, 16, 18, 19, 20, 21, 22, 23, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 42, 44, 45, 47. 48, 49, and/or 50 of Table 2.1, and/or a higher expression of at least one polynucleotide defined by any of the numbers 10, 13, 17, 24, 25, 41, 43, and/or 46, of Table 2.1, is indicative for M4eo when M4eo is distinguished from all other subtypes, and/or wherein a lower expression of at least one polynucleotide defined by any of the numbers 1, 2, 3, 4, 6, 7, 8, 9, 10, 11, 12, 14, 15, 16, 17, 19, 20, 21, 22, 23, 24, 25, 26, 28, 29, 31, 32, 33, 34, 35, 36, 38, 39, 41, 42, 44, 45, 46, 48. 49, and/or 50 of Table 2.2, and/or a higher expression of 5, 13, 18, 27, 30, 37, 40, 43, and/or 47, of Table 2.2 is indicative for PTD when PTD is distinguished from all other subtypes, and/or wherein a lower expression of at least one polynucleotide defined by any of the numbers 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 49, and/or 50 of Table 2.3, and/or a higher expression of at least one polynucleotide defined by any of the numbers 34, and/or 48, of Table 2.3 is indicative for inv3 when inv3 is distinguished from all other subtypes, and/or wherein a lower expression of at least one polynucleotide defined by any of the numbers 1, 2, 3, 5, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 23, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 41, 42, 43, 44, 45, 46, 47, 48, and/or 50 of Table 2.4, and/or a higher expression of at least one polynucleotide defined by any of the numbers 4, 6, 7, 8, 22, 24, 40, and/or 49, of Table 2.4 is indicative for t(15;17) when t(15;17) is distinguished from all other subtypes, and/or wherein a lower expression of at least one polynucleotide defined by any of the numbers 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, and/or 50 of Table 2.5 is indicative for t(8;21) when t(8;21) is distinguished from all other subtypes, and/or wherein a lower expression of at least one polynucleotide defined by any of the numbers 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 13, 14, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 42, 43, 45, 46, 47, 48, 49, and/or 50 of Table 2.6, and/or a higher expression of at least one polynucleotide defined by any of the numbers 12, 15, 29, 41, and/or 44, of Table 2.6 is indicative for tMLL when tMLL is distinguished from all other subtypes, and/or wherein a lower expression of at least one polynucleotide defined by any of the numbers 1, 2, 4, 5, 7, 10, 12, 13, 16, 17, 19, 23, 25, 30, 31, 32, 33, 34, 37, 41, 43, 45, 47, 48, and/or 50 of Table 3.1,and/or a higher expression a polynucleotide defined by any of the numbers 3, 6, 8, 9, 11, 14, 15, 18, 20, 21, 22, 24, 26, 27, 28, 29, 35, 36, 38, 39, 40, 42, 44, 46, and/or 49, of Table 3.1, is indicative for M4eo when M4eo is distinguished from PTD, and/or wherein a lower expression of at least one polynucleotide defined by any of the numbers 5, 6, 9, 12, 23, 28, 38, 41, 44, 45, 46, and/or 47, of Table 3.2, and/or a higher expression of at least one polynucleotide defined by any of the numbers 1, 2, 3, 4, 7, 8, 10, 11, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 24, 25, 26, 27, 29, 30, 31, 32, 33, 34, 35, 36, 37, 39, 40, 42, 43, 48, 49, and/or 50 of Table 3.2, is indicative for M4eo when M4eo is distinguished from inv3, a lower expression of at least one polynucleotide defined by any of the numbers 2, 3, 4, 6, 11, 14, 20, 22, 26, 31, 32, 33, 34, 39, 40, 41, and/or 48, of Table 3.3, and/or a higher expression of at least one polynucleotide defined by any of the numbers 1, 5, 7, 8, 9, 10, 12, 13, 15, 16, 17, 18, 19, 21, 23, 24, 25, 27, 28, 29, 30, 35, 36, 37, 38, 42, 43, 44, 45, 46, 47, 49, and/or 50 of Table 3.3, is indicative for M4eo when M4eo is distinguished from t(15;17), and/or wherein a lower expression of at least one polynucleotide defined by any of the numbers 7, 31, 40, and/or 49, of Table 3.4, and/or a higher expression of at least one polynucleotide defined by any of the numbers 1, 2, 3, 4, 5, 6, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 32, 33, 34, 35, 36, 37, 38, 39, 41, 42, 43, 44, 45, 46, 47, 48, and/or 50 of Table 3.4 is indicative for M4eo when M4eo is distinguished from t(8;21), and/or wherein a lower expression of at least one polynucleotide defined by any of the numbers 1, 3, 10, 14, 17, 18, 19, 21, 24, 25, 26, 31, 32, 34, 41, 44, and/or 50 of Table 3.5, and/or a higher expression of at least one polynucleotide defined by any of the numbers 2, 4, 5, 6, 7, 8, 9, 11, 12, 13, 15, 16, 20, 22, 23, 27, 28, 29, 30, 33, 35, 36, 37, 38, 39, 40, 42, 43, 45, 46, 47, 48, and/or 49, of Table 3.5 is indicative for M4eo when M4eo is distinguished from tMLL, and/or wherein a lower expression of at least one polynucleotide defined by any of the numbers 4, 6, 9, 28, 30, 32, 35, 37, 44, 45, and/or 48, of Table 3.6, and/or a higher expression of at least one polynucleotide defined by any of the numbers 1, 2, 3, 5, 7, 8, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 29, 31, 33, 34, 36, 38, 39, 40, 41, 42, 43, 46, 47, 49, and/or 50 of Table 3.6 is indicative for PTD when PTD is distinguished from inv3, and/or wherein a lower expression of at least one polynucleotide defined by any of the numbers 1, 2, 3, 4, 6, 7, 10, 11, 12, 13, 14, 15, 16, 17, 18, 20, 23, 27, 28, 29, 30, 31, 32, 33, 34, 36, 38, 39, 41, 43, 44, 45, 47, 48, and/or 50 of Table 3.7, and/or a higher expression of polynucleotide defined by any of the numbers 5, 8, 9, 19, 21, 22, 24, 25, 26, 35, 37, 40, 42, 46, and/or 49, of Table 3.7, is for PTD when PTD is distinguished from t(15;17), and/or wherein a lower expression of at least one polynucleotide defined by any of the numbers 7, 9, 10, 11, 13, 16, 20, 21, 22, 23, 30, 35, 36, 38, 42, 45, and/or 50 of Table 3.8, and/or a higher expression of at least one polynucleotide defined by any of the numbers 1, 2, 3, 4, 5, 6, 8, 12, 14, 15, 17, 18, 19, 24, 25, 26, 27, 28, 29, 31, 32, 33, 34, 37, 39, 40, 41, 43, 44, 46, 47, 48, and/or 49, of Table 3.8 is indicative for PTD when PTD is distinguished from t(8;21), and/or wherein a lower expression of at least one polynucleotide defined by any of the numbers 1, 5, 8, 10, 11, 13, 15, 17, 19, 25, 26, 28, 29, 34, and/or 46, of Table 3.9, and/or a higher expression of at least one polynucleotide defined by any of the numbers 2, 3, 4, 6, 7, 9, 12, 14, 16, 18, 20, 21, 22, 23, 24, 27, 30, 31, 32, 33, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 47, 48, 49, and/or 50 of Table 3.9 is indicative for PTD when PTD is distinguished from tMLL, and/or wherein a lower expression of at least one polynucleotide defined by any of the numbers 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 23, 24, 25, 26, 28, 29, 32, 33, 36, 38, 39, 40, 43, 44, 45, 46, 47, and/or 49, of Table 3.10, and/or a higher expression of at least one polynucleotide defined by any of the numbers 22, 27, 30, 31, 34, 35, 37, 41, 42, 48, and/or 50 of Table 3.10, is indicative for inv(3) when inv(3) is distinguished from t(15;17), and/or wherein a lower expression of at least one polynucleotide defined by any of the numbers 1, 5, 6, 9, 11, 12, 15, 17, 18, 19, 23, 27, 35, 36, 37, 39, 42, 43, 47, 49, and/or 50 of Table 3.11, and/or a higher expression of at least one polynucleotide defined by any of the numbers 2, 3, 4, 7, 8, 10, 13, 14, 16, 20, 21, 22, 24, 25, 26, 28, 29, 30, 31, 32, 33, 34, 38, 40, 41, 44, 45, 46, and/or 48, of Table 3.11 is indicative for inv(3) when inv(3) is distinguished from t(8;21), and/or wherein a lower expression of at least one polynucleotide defined by any of the numbers 1, 3, 4, 6, 7, 8, 12, 14, 15, 16, 17, 18, 19, 20, 21, 23, 25, 26, 28, 29, 30, 31, 33, 34, 35, 37, 38, 39, 42, 43, 44, 45, 47, 48, and/or 50 of Table 3.12, and/or a higher expression of at least one polynucleotide defined by any of the numbers 2, 5, 9, 10, 11, 13, 22, 24, 27, 32, 36, 40, 41, 46, and/or 49, of Table 3.12 is indicative for inv(3) when inv(3) is distinguished from tMLL, and/or wherein a lower expression of at least one polynucleotide defined by any of the numbers 3, 4, 7, 14, 16, 20, 22, 23, 24, 25, 26, 30, 35, 36, 37, 39, 40, 43, 44, 46, and/or 50 of Table 3.13, and/or a higher expression of at least one polynucleotide defined by any of the numbers 1, 2, 5, 6, 8, 9, 10, 11, 12, 13, 15, 17, 18, 19, 21, 27, 28, 29, 31, 32, 33, 34, 38, 41, 42, 45, 47, 48, and/or 49 of Table 3.13, is indicative for t(15;17) when t(15;17) is distinguished from t(8;21), and/or wherein a lower expression of at least one polynucleotide defined by any of the numbers 13, 15, 25, 26, 27, 28, 30, 32, 33, 35, 36, 38, 39, 43, 48, and/or 49, of Table 3.14, and/or a higher expression of at least one polynucleotide defined by any of the numbers 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 14, 16, 17, 18, 19, 20, 21, 22, 23, 24, 29, 31, 34, 37, 40, 41, 42, 44, 45, 46, 47, and/or 50 of Table 3.14, is indicative for t(15;17) when t(15;17) is distinguished from tMLL, and/or wherein a lower expression of at least one polynucleotide defined by any of the numbers 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 13, 15, 16, 18, 19, 21, 23, 24, 25, 26, 27, 28, 29, 30, 32, 33, 34, 35, 36, 38, 39, 40, 41, 42, 43, 44, 47, 48, of Table 3.15, and/or a higher expression of at least one polynucleotide defined by any of the numbers 12, 14, 17, 20, 22, 31, 37, 45, 46, 49, and/or 50 of Table 3.15, is indicative for t(8;21) when t(8;21) is distinguished from tMLL.
2. The method according to claim 1 wherein the polynucleotide is labelled.
3. The method according to claim 1 or 2, wherein the label is a luminescent, preferably a fluorescent label, an enzymatic or a radioactive label.
4. The method according at least one of the claims 1-3, wherein the expression level of at least two, preferably of at least ten, more preferably of at least 25, most preferably of 50 of the markers of at least one of the Table 1.1- 3.15 is determined.
5. The method according to at least one of the claims 1-4, wherein the expression level of markers expressed lower in a first subtype than in at least one second subtype, which differs from the first subtype, is at least 5 %, 10% or 20%, more preferred at least 50% or may even be 75% or 100%, i.e. 2-fold lower, preferably at least 10-fold, more preferably at least 50- fold, and most preferably at least 100-fold lower in the first subtype.
6. The method according to at least one of the claims 1-4, wherein the expression level of markers expressed higher in a first subtype than in at least one second subtype, which differs from the first subtype, is at least 5 %, 10% or 20%, more preferred at least 50% or may even be 75% or 100%, i.e. 2-fold higher, preferably at least 10-fold, more preferably at least 50- fold, and most preferably at least 100-fold higher in the first subtype.
7. The method according to at least one of the claims 1-6, wherein the sample is from an individual having AML.
8. The method according to at least one of the claims 1-7, wherein at least one polynucleotide is in the form of a transcribed polynucleotide, or a portion thereof.
9. The method according to claim 8, wherein the transcribed polynucleotide is a rnRNA or a cDNA.
10. The method according to claim 8 or 9, wherein the determining of the expression level comprises hybridizing the transcribed polynucleotide to a complementary polynucleotide, or a portion thereof, under stringent hybridization conditions.
11. The method according to at least one of the claims 1-7, wherein at least one polynucleotide is in the form of a polypeptide, or a portion thereof.
12. The method according to claim 8, 9 or 12, wherein the determining of the expression level comprises contacting the polynucleotide or the polypeptide with a compound specifically binding to the polynucleotide or the polypeptide.
13. The method according to claim 12, wherein the compound is an antibody, or a fragment thereof.
14. The method according to at least one of the claims 1-13, wherein the method is carried out on an array.
15. The method according to at least one of the claims 1-14, wherein the method is carried out in a robotics system.
16. The method according to at least one of the claims 1-15, wherein the method is carried out using microfluidics.
17. Use of at least one marker as defined in at least one of the claims 1-3 for the manufacturing of a diagnostic for distinguishing MLL-PTD-positive AML from other AML subtypes.
18. The use according to claim 17 for distinguishing MLL-PTD-positive AML from other AML subtypes in an individual having AML.
19. A diagnostic kit containing at least one marker as defined in at least one of the claims 1-3 for distinguishing MLL-PTD-positive AML from other AML subtypes, in combination with suitable auxiliaries.
20. The diagnostic kit according to claim 19, wherein the kit contains a reference for the MLL-PTD-positive AML subtypes.
21. The diagnostic kit according to claim 20, wherein the reference is a sample or a data bank.
22. An apparatus for distinguishing MLL-PTD-positive AML from other AML subtypes in a sample containing a reference data bank.
23. The apparatus according to claim 22, wherein the reference data bank is obtainable by comprising (a) compiling a gene expression profile of a patient sample by determining the expression level of at least one marker selected from the markers identifiable by their Affymetrix Identification Numbers (affy id) as defined in Tables 1, 2, 3, 4, 5, 6 and/or 7, and (b) classifying the gene expression profile by means of a machine learning algorithm.
24. The apparatus according to claim 23, wherein the machine learning algorithm is selected from the group consisting of Weighted Noting, K- Νearest Neighbors, Decision Tree Induction, Support Vector Machines, and Feed-Forward Neural Networks, preferably Support Vector Machines.
25. The apparatus according to at least one of the claims 22-24, wherein the apparatus contains a control panel and/or a monitor.
26. A reference data bank for distinguishing MLL-PTD-positive AML from other AML subtypes obtainable by comprising (a) compiling a gene expression profile of a patient sample by determining the expression level of at least one marker selected from the markers identifiable by their Affymetrix Identification Numbers (affy id) as defined in Tables 1, 2, 3, 4, 5, 6 and/or 7, and (b) classifying the gene expression profile by means of a machine learning algorithm.
27. The reference data bank according to claim 26, wherein the reference data bank is backed up and/or contained in a computational memory chip.
PCT/EP2004/012464 2003-11-04 2004-11-04 Method for distinguishing mll-ptd-positive aml from other aml subtypes WO2005043162A2 (en)

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JP2007135467A (en) * 2005-11-17 2007-06-07 National Institute Of Advanced Industrial & Technology UTILIZATION OF Kank 2 GENE FOR CANCER TREATMENT, CANCER DETECTION AND CREATION OF MEDICINE

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