WO2005043161A2 - Method for distinguishing leukemia subtypes - Google Patents

Method for distinguishing leukemia subtypes Download PDF

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Publication number
WO2005043161A2
WO2005043161A2 PCT/EP2004/012463 EP2004012463W WO2005043161A2 WO 2005043161 A2 WO2005043161 A2 WO 2005043161A2 EP 2004012463 W EP2004012463 W EP 2004012463W WO 2005043161 A2 WO2005043161 A2 WO 2005043161A2
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Prior art keywords
value
polynucleotide defined
ofthe numbers
aml
expression
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PCT/EP2004/012463
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French (fr)
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WO2005043161A3 (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 US10/575,635 priority Critical patent/US20070099190A1/en
Priority to EP04797590A priority patent/EP1682901A2/en
Publication of WO2005043161A2 publication Critical patent/WO2005043161A2/en
Publication of WO2005043161A3 publication Critical patent/WO2005043161A3/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 leukemia subtypes, in particular leukemia subtypes AML with t(15;17), t(8;21), inv(16), inv(3), complex aberrant karyotype (CA), AML-MLL, normal karyotype (NK), AML- other, i.e.
  • trisomy 8 trisomy 11, trisomy 13, monosomy 7, del(5q), del(9q), t(6;9); del(20q) and del(12p) and trisomy 4, ALL-MLL, ALL-Ph+, ALL-t(8;14), T-ALL, other B-lineage (OBL), CML, normal-BM (bone marrow), and or CLL, by determining the expression level of selected marker genes.
  • AML acute myeloid
  • ALL acute lymphatic
  • CML chronic myeloid
  • CML chronic lymphatic leukemia
  • 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.
  • 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.
  • 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 ofthe leukemia.
  • CML chronic myeloid leukemia
  • CLL chronic lymphoid
  • 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 CML, the BCR-ABL-rearrangement due to the translocation between chromosomes 9 and 22 (t(9;22) (q34; ql 1)).
  • 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; ql l-12).
  • the introduction of a new drug has improved the outcome in this subgroup of patient from about 50% to 85 % long-term survivors.
  • 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.
  • the technical problem underlying the present invention was to provide means for leukemia diagnostics which overcome at least some ofthe disadvantages of the prior art diagnostic methods, in particular encompassing the time-consxuning and unreliable combination of different methods and which provides a rapid assay to unambigously 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 leukemia subtype from another.
  • the problem is solved by the present invention, which provides a method for distinguishing leukemia subtypes t(15;17), t(8;21), inv(16), inv(3) 5 complex aberrant karyotype (CA), AML-MLL, normal karyotype (NK), AML-other (trisomy 8, trisomy 11, trisomy 13, monosomy 7, del(5q), del(9q), t(6;9); del(20q) and del(12p) and trisomy A), ALL-MLL, ALL-Ph+, ALL-t(8;14), T-ALL, other B- lineage (OBL), CML, normal-BM, and/or CLL 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 and/or 2, wherein a lower expression of at least one polynucleotide defined by at least one ofthe numbers 1 to 50 of Table 1.1
  • a lower expression of at least one polynucleotide defined by at least one ofthe numbers 1 to 50 of Table 2.26 having a negative fc value, and or a higher expression of at least one polynucleotide defined by at least one ofthe numbers 1 to 50 of Table 2.26 having a positive fc value is indicative for the presence ALL_T lineage when ALL_T lineage distinguished from AML_inv(l 6), and or wherein a lower expression of at least one polynucleotide defined by at least one ofthe numbers 1 to 50 of Table 2.27 having a negative fc value, and/or a higher expression of at least one polynucleotide defined by at least one ofthe numbers 1 to 50 of Table 2.27 having a positive fc value, is indicative for the presence ALL_T lineage when ALL_T lineage distinguished from AML_inv(3), and/or wherein a lower expression of at least one polynucleotide defined by at least one ofthe numbers 1 to 50 of
  • t(15;17) AML with t(15;17) translocation
  • t(8;21) AML with t(8;21) translocation inv(16): AML with inversion 16 inv(3): AML with inversion 3
  • CA AML with complex aberrant karyotype
  • AML-MLL AML with mutations on the mixed lineage leukaemia (MLL) gene normal karyotype (NK): AML with normal karyotype trisomy 8: AML with trisomy of chromosome 8 trisomy 11 : AML with trisomy of chromosome 11 trisomy 13: AML with trisomy of chromosome 13 monosomy 7: AML with monosomy of chromosome 7 del(5q): AML with 5q deletion del(9q): AML with 9q deletion t(6;9): AML with t(6;9) translocation del(20q): AML with 20 q deletion del(12p): AML with deletion 12 p deletion trisomy 4: AML with trisomy 4
  • ALL-MLL acute lymphoblastic leukaemia with mutations on the mixed lineage leukemia (MLL) gene
  • ALL-Ph+ acute lymphoblastic leukaemia with genetic aberration on the Philadelphia chromosome
  • ALL-t(8;14) acute lymphoblastic leukemia with translocation t(8;21)
  • T-ALL T cell acute lymphoblastic leukemia other B-lineage (OBL):
  • CLL chronic lymphatic leukemia
  • all other subtypes refer to the subtypes of the present invention, i.e. to all other subtypes except for the one being under investigation
  • 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.
  • 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.
  • 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-antibody- 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 inco ⁇ orated into, e.g. the cRNA polynucleotide by in vitro transcription.
  • cDNA may be prepared into which a detectable label, as exemplified above, is inco ⁇ orated. 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 counte ⁇ art 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 inco ⁇ orated 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 Harlow and Lane, "Antibodies, a laboratory manual", CSH Press, 1988, Cold
  • a minimum set of proteins necessary for diagnosis of all leukemia 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 microtite ⁇ lates.
  • 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 immunoassay.
  • CA complex aberrant karyotype
  • AML-MLL normal karyotype
  • NK normal karyotype
  • AML- other i.e. trisomy 8 trisomy 11, trisomy 13, monosomy 7, del(5q), del(9q), t(6
  • the statistical significance of markers as expressed in q or p values based on the concept ofthe false discovery rate is determined.
  • 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 ofthe false discovery rate rather than the false positive rate (Storey JD and Tibsbirani R. Proc.Natl.Acad.Sci., 2003, Vol. 100:9440-5.
  • the expression level of at least two, preferably of at least ten, more preferably of at least 25, most preferably of 50 of at least one ofthe Tables ofthe markers is determined.
  • 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 ofthe Tables are measured.
  • the level of the expression of the handheldmarker i.e. the expression of the polynucleotide is indicative of the leukemia 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 ofthe 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 preferred 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 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.
  • 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 leukemia.
  • 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.
  • 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 ofthe 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 (lower percentages of formamide result in lowered stringency), salt conditions, or temperature.
  • 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 ofthe 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 ofthe 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.
  • Preferred 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 resoance energy transfer between two adjacenly hybridized probes as used in the LightCycler® format described in U.S.
  • Example 3 A preferred 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 inco ⁇ orated during the transcription reaction.
  • the purified cRNA is applied to commercially available arrays which can be obtained e.g. from Affymetrix.
  • the hybridized cRNA is detected according to the methods described in Example 3.
  • the arrays 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 0 373 203, 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 ofthe polynucleotides or polypeptides is detected using a compound which specifically binds to the polynucleotide ofthe polypeptide ofthe 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 K ⁇ hler 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 immuno sorbent assay (RIA), dissociation-enhanced lanthanide fluoro immuno assay (DELFIA), scintillation proximity assay (SPA).
  • ELISA Enzyme-linked immuno sorbent assay
  • RIA Enzyme-linked immuno sorbent assay
  • DELFIA dissociation-enhanced lanthanide fluoro immuno assay
  • SPA scintillation proximity assay
  • an "array” or “microarray” refers to a linear or two- or three dimensional arrangement 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 "array”.
  • a microarray usually refers to a miniaturised array arrangement, with the probes being attached to a density of at least about 10, 20, 50, 100 nucleic acid molecules referring to different or the same genes per cm 2 .
  • an array can be referred to as "gene chip”.
  • the array 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 array fabrication is well-known to the person skilled in the art.
  • the process for preparing a nucleic acid array 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 information of custom cDNA libraries or to identify cDNA clones from previously prepared libraries.
  • the liquid containing the amplified probes can be deposited on the array 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 array.
  • the array is a high density oligonucleotide (oligo) array using a light-directed chemical synthesis process, employing the so-called photolithography technology.
  • oligo arrays are a high density oligonucleotide (oligo) array using a light-directed chemical synthesis process, employing the so-called photolithography technology.
  • oligo arrays are a high density oligonucleotide (oligo) array using a light-directed chemical synthesis process, employing the so-called photolithography technology.
  • oligo arrays Unlike common cDNA arrays, oligo arrays
  • the sequence can be synthesized directly onto the array, thus, bypassing the need for physical intermediates, such as PCR products, required for making cDNA arrays.
  • 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 carried out in a robotics system including robotic plating and a robotic liquid transfer system, e.g. using microfluidics, i.e. channelled structured.
  • a particular preferred 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 Numbers (affy id) as defined in Tables 1, and/or 2 for the manufacturing of a diagnostic for distinguishing Leukemia subtypes.
  • the use of the present invention is particularly advantageous for distinguishing leukemia subtypes in an individual having leukemia.
  • markers for diagnosis of leukemia subtypes preferably based on microarray 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 cytomo ⁇ hology 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 2 for distinguishing leukemia 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 corresponding 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 t(15;17), t(8;21), inv(16), inv(3), complex aberrant karyotype (CA), AML- MLL, normal karyotype (NK), AML-other, i.e. trisomy 8, trisomy 11, trisomy 13, monosomy 7, del(5q), del(9q), t(6;9); del(20q) and del(12p) and trisomy 4, ALL-
  • the reference can be a sample or a data bank.
  • the present invention is directed to an apparatus for distinguishing leukemia subtypes t(15;17), t(8;21), inv(16), inv(3), complex aberrant karyotype (CA), AML-MLL, normal karyotype (NK), AML-other, i.e.
  • 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 2, and (b) classifying the gene expression profile by means of a machine learning algorithm.
  • 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 corresponding to a certain expression level which are obtained by the microarray 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 microarray 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 referred 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.
  • the classification accuracy of a given gene list for a set of microarray 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 correct predictions of the complete data set was estimated by lOfold cross validation.
  • SVM-model ofthe training set was built to predict diagnosis in the independent test set, thereby estimating true accuracy of the prediction model.
  • 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 ofthe present invention, e.g. like an interchangeable module, in order to use another data memory chip containing a different reference data bank.
  • the apparatus ofthe 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 microarray 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 ofthe 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 ofthe 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 inco ⁇ orated 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 microarray containing the hybridized nucleic acids.
  • the present invention refers to a reference data bank for extinguishing leukemia subtypes t(15;17), t(8;21), inv(16), inv(3), complex aberrant karyotype (CA), AML-MLL, normal karyotype (NK), AML-other, i.e.
  • trisomy 8 trisomy 11, trisomy 13, monosomy 7, del(5q), del(9q), t(6;9); del(20q) and del(12p) and trisomy 4, ALL-MLL, ALL-Ph+, ALL-t(8;14), T-ALL, other B- lineage (OBL), CML, normal-BM, and/or CLL 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 7, and (b) classifying the gene expression profile by means of a machine learning algorithm.
  • affymetrix Identification Numbers as defined in Tables 1, and/or 7
  • the reference data bank is backed up and/or contained in a computational memory data chip.
  • Tables 1.1 to 2.78 show leukemia subtype analysis of t(15;17), t(8;21), inv(16), inv(3), complex aberrant karyotype (CA), AML-MLL, normal karyotype (NK), AML-other, i.e. trisomy 8, trisomy 11, trisomy 13, monosomy 7, del(5q), del(9q), t(6;9); del(20q) and del(12p) and trisomy 4, ALL-MLL, ALL-Ph+, ALL-t(8;14), T- ALL, other B-lineage (OBL), CML, normal-BM, and/or CLL.
  • the analysed markers are ordered according to their q- values, beginning with the lowest q- values.
  • Tables 1.1 to 2.78 are accompanied with explanatory tables (Table 1.1 A to 2.78A) where the numbering and the Affymetrix Id are further defined by other parameters, e.g. gene bank accession number
  • Example 1 General experimental design of the invention and results So far comprehensive diagnosis of leukemia requires a combination of cytomo ⁇ hology, immunophenotyping, and genetic methods. We aimed at developing a diagnostic tool based only on gene expression profiling to accurately predict all clinically relevant subtypes of leukemia. Therefore, we analyzed samples from 540 patients at diagnosis using oligonucleotide microarrays representing 33,000 different genes (U133 set, Affymetrix).
  • OBL B-lineage
  • To identify differentially expressed genes we applied ANOVA and t-test-statistics (Welch t- test).
  • To assess the false discovery rate we calculated q values according to Storey et al. (PNAS, 2003).
  • To estimate diagnostic accuracy based on gene expression signatures we designated a training set consisting of 2/3 of cases and a test set with 1/3 of cases. Assignment of cases to training and test set was randomized and balanced by diagnosis. Based on the fraining set we built Support Vector Machine (SVM) models. Classification accuracy was assessed in the independent test set. In the first analysis five main categories AML, ALL, CML, CLL, and n-BM were distinguished in the test set with an accuracy of 96% (177/184 correctly assigned). In a second analysis the following 13 subtypes were
  • the methods section contains both information on statistical analyses used for identification of differentially expressed genes and detailed annotation data of identified microarray 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 Center (www.affymetrix.com)
  • Microarray probesets for example found to be differentially expressed between different types of leukemia samples are further described by additional information.
  • HG-U133 ProbeSet D describes the probe set identifier. Examples are:
  • 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 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.
  • 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.
  • GenBank® GenBank®, RefSeq, UniGene, TIGR (annotations from The Institute for Genomic Research).
  • 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
  • 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
  • This information represents the LocusLink accession number.
  • Example 3 Sample preparation, processing and data analysis
  • Microarray 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,
  • RNA samples were thawed, homogenized (QIAshredder, Qiagen), and total RNA was extracted (RNeasy Mini Kit, Qiagen). Subsequently, 5-10 ⁇ g total RNA isolated from 1 x IO 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 inco ⁇ orated during the following in vitro transcription reaction (Enzo BioArray HighYield
  • RNA Transcript Labeling Kit Enzo Diagnostics
  • 15 ⁇ g 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 microarrays (300 ⁇ l final volume). Washing and staining of the probe arrays was performed according to the recommended Fluidics Station protocol (EukGE-WS2v4).
  • Affymetrix Microarray Suite software (version 5.0.1) extracted fluorescence signal intensities from each feature on the microarrays as detected by confocal laser scanning according to the manufacturer's recommendations.
  • Expression analysis quality assessment parameters included visiual array inspection ofthe scanned image for the presence of image artifacts and correct 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 ofthe 3' probe sets for GAPDH are compared to the Signal values of the corresponding 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 Microarray Analysis Suite MAS 5.0 software package.
  • Bone marrow (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 GeneChip System (Affymetrix, Santa Clara, CA, USA) is used.
  • the targets for GeneChip analysis are prepared according to the current Expression Analysis.
  • RNA extracted RNA extracted 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.
  • biotin-labeled ribonucleotides are inco ⁇ orated during the in vitro transcription reaction (Enzo® Bio ArrayTM
  • RNA Transcript Labeling Kit ENZO
  • 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 microarrays.
  • Test3 Probe Arrays 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 arrays (Affymetrix). Washing and staining the Probe arrays is performed as described ( founded Affymetrix-Original- Literatur (LOCKHART und LIPSHUTZ). The Affymetrix software (Microarray Suite, Version 4.0.1) extracted fluorescence intensities from each element on the arrays as detected by confocal laser scanning according to the manufacturers recommendations.
  • OVA One-Versus-AII

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Abstract

Disclosed is a method for distinguishing leukemia subtypes t(15;17), t(8,21), inv(16), inv(3), complex aberrant karyotype (CA), AML-MLL, normal karotype (NK), AML-other (trisomy 8, trisomy 11, trisomy 13, monosomy 7, del(5q), del(9q), t(6;9); del (20q) and del(12p) and trisomy 4), ALL-MLL, ALL Ph+, ALL-t(8, 14), T-ALL, other B-lineage (OBL), CML, normal-BM, and/or CLL 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 leukemia subtypes
The present invention is directed to a method for distinguishing leukemia subtypes, in particular leukemia subtypes AML with t(15;17), t(8;21), inv(16), inv(3), complex aberrant karyotype (CA), AML-MLL, normal karyotype (NK), AML- other, i.e. trisomy 8, trisomy 11, trisomy 13, monosomy 7, del(5q), del(9q), t(6;9); del(20q) and del(12p) and trisomy 4, ALL-MLL, ALL-Ph+, ALL-t(8;14), T-ALL, other B-lineage (OBL), CML, normal-BM (bone marrow), and or CLL, 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 subcatgories 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 ofthe 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 lymphoid (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 ofthe 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) 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; ql 1)). 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; ql l-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 ofthe disadvantages of the prior art diagnostic methods, in particular encompassing the time-consxuning and unreliable combination of different methods and which provides a rapid assay to unambigously 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 ofthe two different subgroups was performed using exclusively mo hologic-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 leukemia subtype from another.
The problem is solved by the present invention, which provides a method for distinguishing leukemia subtypes t(15;17), t(8;21), inv(16), inv(3)5 complex aberrant karyotype (CA), AML-MLL, normal karyotype (NK), AML-other (trisomy 8, trisomy 11, trisomy 13, monosomy 7, del(5q), del(9q), t(6;9); del(20q) and del(12p) and trisomy A), ALL-MLL, ALL-Ph+, ALL-t(8;14), T-ALL, other B- lineage (OBL), CML, normal-BM, and/or CLL 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 and/or 2, wherein a lower expression of at least one polynucleotide defined by at least one ofthe numbers 1 to 50 of Table 1.1 having a negative fc value, and/or a higher expression of at least one polynucleotide defined by at least one ofthe numbers 1 to 50 of Table 1.1 having apositive fc value, is indicative for the presence ALLJVILL when ALL_MLL is distinguished from all other subtypes, and/or wherein a lower expression of at least one polynucleotide defined by at least one ofthe numbers 1 to 50 of Table 1.2 having a negative fc value, and/or a higher expression of at least one polynucleotide defined by at least one ofthe numbers 1 to 50 of Table 1.2 having a positive fc value, is indicative for the presence ALL_Ph+ when ALL_Ph+ is distinguished from all other subtypes, and/or wherein a lower expression of at least one polynucleotide defined by at least one ofthe numbers 1 to 50 of Table 1.3 having a negative fc value, and/or a higher expression of at least one polynucleotide defined by at least one ofthe numbers 1 to 50 of Table 1.3 having a positive fc value, is indicative for the presence ALL_T lineage when ALL_T lineage is distinguished from all other subtypes, and/or wherein a lower expression of at least one polynucleotide defined by at least one ofthe numbers 1 to 50 of Table 1.4 having a negative fc value, and/or a higher expression of at least one polynucleotide defined by at least one ofthe numbers 1 to 50 of Table 1.4 having a positive fc value, is indicative for the presence ALL_t(8;14) when ALL_t(8;14) is distinguished from all other subtypes, and/or wherein a lower expression of at least one polynucleotide defined by at least one ofthe numbers 1 to 50 of Table 1.5 having a negative fc value, and/or a higher expression of at least one polynucleotide defined by at least one ofthe numbers 1 to 50 of Table 1.5 having a positive fc value, is indicative for the presence AML_MLL when AMLJVΪLL is distinguished from all other subtypes, and/or wherein a lower expression of at least one polynucleotide defined by at least one ofthe numbers 1 to 50 of Table 1.6 having a negative fc value, and/or a higher expression of at least one polynucleotide defined by at least one ofthe numbers 1 to 50 of Table 1.6 having a positive fc value, is indicative for the presence AML_inv(l 6) when AML_inv(l 6) is distinguished from all other subtypes, and/or wherein a lower expression of at least one polynucleotide defined by at least one ofthe numbers 1 to 50 of Table 1.7 having a negative fc value, and/or a higher expression of at least one polynucleotide defined by at least one ofthe numbers 1 to 50 of Table 1.7 having a positive fc value, is indicative for the presence AML_inv(3) when AML_inv(3) is distinguished from all other subtypes, and/or wherein a lower expression of at least one polynucleotide defined by at least one ofthe numbers 1 to 50 of Table 1.8 having a negative fc value, and/or a higher expression of at least one polynucleotide defined by at least one ofthe numbers 1 to 50 of Table 1.8 having a positive fc value, is indicative for the presence AML_komplext when AML_komplext is distinguished from all other subtypes, and/or wherein a lower expression of at least one polynucleotide defined by at least one ofthe numbers 1 to 50 of Table 1.9 having a negative fc value, and/or a higher expression of at least one polynucleotide defined by at least one ofthe numbers 1 to 50 of Table 1.9 having a positive fc value, is indicative for the presence AML_t(15;17) when AML_t(15;17) is distinguished from all other subtypes, and/or wherein a lower expression of at least one polynucleotide defined by at least one ofthe numbers 1 to 50 of Table 1.10 having a negative fc value, and/or a higher expression of at least one polynucleotide defined by at least one ofthe numbers 1 to 50 of Table 1.10 having a positive fc value, is indicative for the presence AML_t(8;21) when AML_t(8;21) is distinguished from all other subtypes, and/or wherein a lower expression of at least one polynucleotide defined by at least one ofthe numbers 1 to 50 of Table 1.11 having a negative fc value, and/or a higher expression of at least one polynucleotide defined by at least one ofthe numbers 1 to 50 of Table 1.11 having a positive fc value, is indicative for the presence CLL when CLL is distinguished from all other subtypes, and/or wherein a lower expression of at least one polynucleotide defined by at least one ofthe numbers 1 to 50 of Table 1.12 having a negative fc value, and/or a higher expression of at least one polynucleotide defined by at least one ofthe numbers 1 to 50 of Table 1.12 having a positive fc value, is indicative for the presence CML when CML is distinguished from all other subtypes, and/or wherein a lower expression of at least one polynucleotide defined by at least one ofthe numbers 1 to 50 of Table 1.13 having a negative fc value, and/or a higher expression of at least one polynucleotide defined by at least one ofthe numbers 1 to 50 of Table 1.13 having a positive fc value, is indicative for the presence normal-BM when normal-BM is distinguished from all leukemia subtypes, and/or wherein a lower expression of at least one polynucleotide defined by at least one ofthe numbers 1 to 50 of Table 2.1 having a negative fc value, and/or a higher expression of at least one polynucleotide defined by at least one ofthe numbers 1 to 50 of Table 2.1 having a positive fc value, is indicative for the presence ALL_MLL when ALL_MLL is distinguished from ALL_Ph+, and/or wherein a lower expression of at least one polynucleotide defined by at least oae ofthe numbers 1 to 50 of Table 2.2 having a negative fc value, and/or a higher expression of at least one polynucleotide defined by at least one ofthe numbers 1 to 50 of Table 2.2 having a positive fc value, is indicative for the presence ALL_MLL when ALL_MLL is distinguished from ALL_T lineage, and/or wherein a lower expression of at least one polynucleotide defined by at least one ofthe numbers 1 to 50 of Table 2.3 having a negative fc value, and/or a higher expression of at least one polynucleotide defined by at least one ofthe numbers 1 to 50 of Table 2.3 having a positive fc value, is indicative for the presence ALL_MLL when ALL_MLL is distinguished from ALL_t(8; 14), and/or wherein a lower expression of at least one polynucleotide defined by at least one ofthe numbers 1 to 50 of Table 2.4 having a negative fc value, and/or a higher expression of at least one polynucleotide defined by at least one ofthe numbers 1 to 50 of Table 2.4 having a positive fc value, is indicative for the presence ALL_MLL when ALLJVILL is distinguished from AML_MLL, and/or wherein a lower expression of at least one polynucleotide defined by at least one ofthe numbers 1 to 50 of Table 2.5 having a negative fc value, and/or a higher expression of at least one polynucleotide defined by at least one ofthe numbers 1 to 50 of Table 2.5 having a positive fc value, is indicative for the presence ALLJVILL when ALLJVILL is distinguished from AML_inv(16), and/or wherein a lower expression of at least one polynucleotide defined by at least one ofthe numbers 1 to 50 of Table 2.6 having a negative fc value, and or a higher expression of at least one polynucleotide defined by at least one ofthe numbers 1 to 50 of Table 2.6 having a positive fc value, is indicative for the presence ALLJVILL when ALLJVILL is distinguished from AML_inv(3), and/or wherein a lower expression of at least one polynucleotide defined by at least one ofthe numbers 1 to 50 of Table 2.7 having a negative fc value, and/or a higher expression of at least one polynucleotide defined by at least one ofthe numbers 1 to 50 of Table 2.7 having a positive fc value, is indicative for the presence ALLJVILL when ALLJVILL is distinguished from AML_komplext, and/or wherein a lower expression of at least one polynucleotide defined by at least one ofthe numbers 1 to 50 of Table 2.8 having a negative fc value, and/or a higher expression of at least one polynucleotide defined by at least one ofthe numbers 1 to 50 of Table 2.8 having a positive fc value, is indicative for the presence ALLJVILL when ALLJVILL is distinguished from AML_t(l 5; 17), and/or wherein a lower expression of at least one polynucleotide defined by at least one ofthe numbers 1 to 50 of Table 2.9 having a negative fc value, and/or a higher expression of at least one polynucleotide defined by at least one ofthe numbers 1 to 50 of Table 2.9 having a positive fc value, is indicative for the presence ALLJVILL when ALLJVILL is distinguished from AML_t(8;21), and/or wherein a lower expression of at least one polynucleotide defined by at least one ofthe numbers 1 to 50 of Table 2.10 having a negative fc value, and/or a higher expression of at least one polynucleotide defined by at least one ofthe numbers 1 to 50 of Table 2.10 having a positive fc value, is indicative for the presence ALLJVILL when ALLJVILL is distinguished from CLL, and/or wherein a lower expression of at least one polynucleotide defined by at least one ofthe numbers 1 to 50 of Table 2.11 having a negative fc value, and/or a higher expression of at least one polynucleotide defined by at least one ofthe numbers 1 to 50 of Table 2.11 having a positive fc value, is indicative for the presence ALLJVILL when ALLJVILL is distinguished from CML, and/or wherein a lower expression of at least one polynucleotide defined by at least one ofthe numbers 1 to 50 of Table 2.12 having a negative fc value, and/or a higher expression of at least one polynucleotide defined by at least one ofthe numbers 1 to 50 of Table 2.12 having a positive fc value, is indicative for the presence ALLJVILL when ALLJVILL is distinguished from normal-BM, and or wherein a lower expression of at least one polynucleotide defined by at least one ofthe numbers 1 to 50 of Table 2.13 having a negative fc value, and/or a higher expression of at least one polynucleotide defined by at least one ofthe numbers 1 to 50 of Table 2.13 having a positive fc value, is indicative for the presence ALL_Ph+ when ALLJPh+ is distinguished from ALL_T lineage, and/or wherein a lower expression of at least one polynucleotide defined by at least one ofthe numbers 1 to 50 of Table 2.14 having a negative fc value, and or a higher expression of at least one polynucleotide defined by at least one ofthe numbers 1 to 50 of Table 2.14 having a positive fc value, is indicative for the presence ALL_Ph+ when ALL_Ph+ is distinguished from ALL_t(8;14), and/or wherein a lower expression of at least one polynucleotide defined by at least one ofthe numbers 1 to 50 of Table 2.15 having a negative fc value, and or a higher expression of at least one polynucleotide defined by at least one ofthe numbers 1 to 50 of Table 2.15 having a positive fc value, is indicative for the presence ALL_Ph+ when ALL_Ph+ is distinguished from AMLJVILL, and/or wherein a lower expression of at least one polynucleotide defined by at least one ofthe numbers 1 to 50 of Table 2.16 having a negative fc value, and or a higher expression of at least one polynucleotide defined by at least one ofthe numbers 1 to 50 of Table 2.16 having a positive fc value, is indicative for the presence ALL_Ph+ when ALL_Ph+ is distinguished from AML_inv( 16), and/or wherein a lower expression of at least one polynucleotide defined by at least one ofthe numbers 1 to 50 of Table 2.17 having a negative fc value, and/or a higher expression of at least one polynucleotide defined by at least one ofthe numbers 1 to 50 of Table 2.17 having a positive fc value, is indicative for the presence ALLJPh+ when ALLJPh+ is distinguished from AML_inv(3), and/or wherein a lower expression of at least one polynucleotide defined by at least one ofthe numbers 1 to 50 of Table 2.18 having a negative fc value, and/or a higher expression of at least one polynucleotide defined by at least one ofthe numbers 1 to 50 of Table 2.18 having a positive fc value, is indicative for the presence ALLJPh÷ when ALL_Ph+ is distinguished from AMLJcomplext, and/or wherein a lower expression of at least one polynucleotide defined by at least one ofthe numbers 1 to 50 of Table 2.19 having a negative fc value, and/or a higher expression of at least one polynucleotide defined by at least one ofthe numbers 1 to 50 of Table 2.19 having a positive fc value, is indicative for the presence ALL_Ph+ when ALL_Ph+ is distinguished from AML_t(l 5; 17), and/or wherein a lower expression of at least one polynucleotide defined by at least one ofthe numbers 1 to 50 of Table 2.20 having a negative fc value, and/or a higher expression of at least one polynucleotide defined by at least one ofthe numbers 1 to 50 of Table 2.20 having a positive fc value, is indicative for the presence ALL_Ph+ when ALL_Ph+ is distinguished from AML_t(8;21), and/or wherein a lower expression of at least one polynucleotide defined by at least one ofthe numbers 1 to 50 of Table 2.21 having a negative fc value, and/or a higher expression of at least one polynucleotide defined by at least one ofthe numbers 1 to 50 of Table 2.21 having a positive fc value, is indicative for the presence ALL_Ph+ when ALL_Ph+ is distinguished from CLL, and/or wherein a lower expression of at least one polynucleotide defined by at least one ofthe numbers 1 to 50 of Table 2.22 having a negative fc value, and/or a higher expression of at least one polynucleotide defined by at least one ofthe numbers 1 to 50 of Table 2.22 having a positive fc value, is indicative for the presence ALL_Ph+ when ALL_Ph+ is distinguished from CML, and/or wherein a lower expression of at least one polynucleotide defined by at least one ofthe numbers 1 to 50 of Table 2.23 having a negative fc value, and/or a higher expression of at least one polynucleotide defined by at least one ofthe numbers 1 to 50 of Table 2.23 having a positive fc value, is indicative for the presence ALL_Ph+ when ALL_Ph+ is distinguished from normal-BM, and/or wherein a lower expression of at least one polynucleotide defined by at least one ofthe numbers 1 to 50 of Table 2.24 having a negative fc value, and or a higher expression of at least one polynucleotide defined by at least one ofthe numbers 1 to 50 of Table 2.24 having a positive fc value, is indicative for the presence ALL_T lineage when ALL_T lineage distinguished from ALL_t(8;14), and/or wherein a lower expression of at least one polynucleotide defined by at least one ofthe numbers 1 to 50 of Table 2.25 having a negative fc value, and/or a higher expression of at least one polynucleotide defined by at least one ofthe numbers 1 to 50 of Table 2.25 having a positive fc value, is indicative for the presence ALL_T lineage when ALL_T lineage distinguished from AMLJVILL,
•and/or wherein a lower expression of at least one polynucleotide defined by at least one ofthe numbers 1 to 50 of Table 2.26 having a negative fc value, and or a higher expression of at least one polynucleotide defined by at least one ofthe numbers 1 to 50 of Table 2.26 having a positive fc value, is indicative for the presence ALL_T lineage when ALL_T lineage distinguished from AML_inv(l 6), and or wherein a lower expression of at least one polynucleotide defined by at least one ofthe numbers 1 to 50 of Table 2.27 having a negative fc value, and/or a higher expression of at least one polynucleotide defined by at least one ofthe numbers 1 to 50 of Table 2.27 having a positive fc value, is indicative for the presence ALL_T lineage when ALL_T lineage distinguished from AML_inv(3), and/or wherein a lower expression of at least one polynucleotide defined by at least one ofthe numbers 1 to 50 of Table 2.28 having a negative fc value, and/or a higher expression of at least one polynucleotide defined by at least one ofthe numbers 1 to 50 of Table 2.28 having a positive fc value, is indicative for the presence ALL_T lineage when ALL_T lineage distinguished from AML_komplext, and/or wherein a lower expression of at least one polynucleotide defined by at least one ofthe numbers 1 to 50 of Table 2.29 having a negative fc value, and/or a higher expression of at least one polynucleotide defined by at least one ofthe numbers 1 to 50 of Table 2.29 having a positive fc value, is indicative for the presence ALL_T lineage when ALL_T lineage distinguished from AML_t( 15 ; 17), and/or wherein a lower expression of at least one polynucleotide defined by at least one ofthe numbers 1 to 50 of Table 2.30 having a negative fc value, and/or a higher expression of at least one polynucleotide defined by at least one ofthe numbers 1 to 50 of Table 2.30 having a positive fc value, is indicative for the presence ALL_T lineage when ALL_T lineage distinguished from AML_t(8;21), and/or wherein a lower expression of at least one polynucleotide defined by at least one ofthe numbers 1 to 50 of Table 2.31 having a negative fc value, and/or a higher expression of at least one polynucleotide defined by at least one ofthe numbers 1 to 50 of Table 2.31 having a positive fc value, is indicative for the presence ALL_T lineage when ALL_T lineage distinguished from CLL, and/or wherein a lower expression of at least one polynucleotide defined by at least one ofthe numbers 1 to 50 of Table 2.32 having a negative fc value, and/or a higher expression of at least one polynucleotide defined by at least one ofthe numbers 1 to 50 of Table 2.32 having a positive fc value, is indicative for the presence ALL_T lineage when ALL_T lineage distinguished from CML, and/or wherein a lower expression of at least one polynucleotide defined by at least one ofthe numbers 1 to 50 of Table 2.33 having a negative fc value, and/or a higher expression of at least one polynucleotide defined by at least one ofthe numbers 1 to 50 of Table 2.33 having a positive fc value, is indicative for the presence ALL_T lineage when ALL_T lineage distinguished from normal-BM, and/or wherein a lower expression of at least one polynucleotide defined by at least one ofthe numbers 1 to 50 of Table 2.34 having a negative fc value, and/or a higher expression of at least one polynucleotide defined by at least one ofthe numbers 1 to 50 of Table 2.34 having a positive fc value, is indicative for the presence ALL_t(8;14) when ALL_t(8;14) distinguished from AML_MLL, andor wherein a lower expression of at least one polynucleotide defined by at least one ofthe numbers 1 to 50 of Table 2.35 having a negative fc value, and/or a higher expression of at least one polynucleotide defined by at least one ofthe numbers 1 to 50 of Table 2.35 having a positive fc value, is indicative for the presence ALL_t(8;14) when ALL_t(8;14) distinguished from AML_inv(16), and/or wherein a lower expression of at least one polynucleotide defined by at least one ofthe numbers 1 to 50 of Table 2.36 having a negative fc value, and/or a higher expression of at least one polynucleotide defined by at least one ofthe numbers 1 to 50 of Table 2.36 having a positive fc value, is indicative for the presence ALL_t(8;14) when ALL_t(8;14) distinguished from AML_inv(3), and/or wherein a lower expression of at least one polynucleotide defined by at least one ofthe numbers 1 to 50 of Table 2.37 having a negative fc value, and/or a higher expression of at least one polynucleotide defined by at least one ofthe numbers 1 to 50 of Table 2.37 having a positive fc value, is indicative for the presence ALL_t(8 ; 14) when ALL_t(8 ; 14) distinguished from AML_komplext, and/or wherein a lower expression of at least one polynucleotide defined by at least one ofthe numbers 1 to 50 of Table 2.38 having a negative fc value, and/or a higher expression of at least one polynucleotide defined by at least one ofthe numbers 1 to 50 of Table 2.38 having a positive fc value, is indicative for the presence ALL_t(8;14) when ALL_t(8;14) distinguished from AML_t(15;17), and/or wherein a lower expression of at least one polynucleotide defined by at least one ofthe numbers 1 to 50 of Table 2.39 having a negative fc value, and/or a higher expression of at least one polynucleotide defined by at least one ofthe numbers 1 to 50 of Table 2.39 having a positive fc value, is indicative for the presence ALL_t(8;14) when ALL_t(8;14) distinguished from AML_t(8;21), and/or wherein a lower expression of at least one polynucleotide defined by at least one ofthe numbers 1 to 50 of Table 2.40 having a negative fc value, and/or a higher expression of at least one polynucleotide defined by at least one ofthe numbers 1 to 50 of Table 2.40 having a positive fc value, is indicative for the presence ALL_t(8;14) when ALL_t(8;14) distinguished from CLL, and/or wherein a lower expression of at least one polynucleotide defined by at least one ofthe numbers 1 to 50 of Table 2.41 having a negative fc value, and/or a higher expression of at least one polynucleotide defined by at least one ofthe numbers 1 to 50 of Table 2.41 having a positive fc value, is indicative for the presence ALL_t(8;14) when ALL_t(8;14) distinguished from CML, and/or wherein a lower expression of at least one polynucleotide defined by at least one ofthe numbers 1 to 50 of Table 2.42 having a negative fc value, and/or a higher expression of at least one polynucleotide defined by at least one ofthe numbers 1 to 50 of Table 2.42 having a positive fc value, is indicative for the presence ALL_t(8;14) when ALL_t(8;14) distinguished from normal-BM, and/or wherein a lower expression of at least one polynucleotide defined by at least one ofthe numbers 1 to 50 of Table 2.43 having a negative fc value, and/or a higher expression of at least one polynucleotide defined by at least one ofthe numbers 1 to 50 of Table 2.43 having a positive fc value, is indicative for the presence AMLJV1LL when AMLJVILL distinguished from AML_inv(16), and/or wherein a lower expression of at least one polynucleotide defined by at least one ofthe numbers 1 to 50 of Table 2.44 having a negative fc value, and/or a higher expression of at least one polynucleotide defined by at least one ofthe numbers 1 to 50 of Table 2.44 having a positive fc value, is indicative for the presence AMLJVILL when AML_MLL distinguished from AML_inv(3), and/or wherein a lower expression of at least one polynucleotide defined by at least one ofthe numbers 1 to 50 of Table 2.45 having a negative fc value, and/or a higher expression of at least one polynucleotide defined by at least one ofthe numbers 1 to 50 of Table 2.45 having a positive fc value, is indicative for the presence AMLJVILL when AMLJVILL distinguished from AML_komplext, and/or wherein a lower expression of at least one polynucleotide defined by at least one ofthe numbers 1 to 50 of Table 2.46 having a negative fc value, and or a higher expression of at least one polynucleotide defined by at least one ofthe numbers 1 to 50 of Table 2.46 having a positive fc value, is indicative for the presence AMLJVILL when AMLJVILL distinguished from AML_t(15;17), and/or wherein a lower expression of at least one polynucleotide defined by at least one ofthe numbers 1 to 50 of Table 2.47 having a negative fc value, and/or a higher expression of at least one polynucleotide defined by at least one ofthe numbers 1 to 50 of Table 2.47 having a positive fc value, is indicative for the presence AMLJVILL when AMLJVILL distinguished from AML_t(8 ;21 ), and/or wherein a lower expression of at least one polynucleotide defined by at least one ofthe numbers 1 to 50 of Table 2.48 having a negative fc value, and/or a higher expression of at least one polynucleotide defined by at least one ofthe numbers 1 to 50 of Table 2.48 having a positive fc value, is indicative for the presence AMLJVILL when AMLJVILL distinguished from CLL, and/or wherein a lower expression of at least one polynucleotide defined by at least one ofthe numbers 1 to 50 of Table 2.49 having a negative fc value, and/or a higher expression of at least one polynucleotide defined by at least one ofthe numbers 1 to 50 of Table 2.49 having a positive fc value, is indicative for the presence AMLJVILL when AMLJVILL distinguished from CML, and/or wherein a lower expression of at least one polynucleotide defined by at least one ofthe numbers 1 to 50 of Table 2.50 having a negative fc value, and or a higher expression of at least one polynucleotide defined by at least one ofthe numbers 1 to 50 of Table 2.50 having a positive fc value, is indicative for the presence AMLJVILL when AMLJVILL distinguished from normal-BM, and/or wherein a lower expression of at least one polynucleotide defined by at least one ofthe numbers 1 to 50 of Table 2.51 having a negative fc value, and/or a higher expression of at least one polynucleotide defined by at least one ofthe numbers 1 to 50 of Table 2.51 having a positive fc value, is indicative for the presence AML_inv(16) when AML_inv(16) distinguished from AML_inv(3), and/or wherein a lower expression of at least one polynucleotide defined by at least one ofthe numbers 1 to 50 of Table 2.52 having a negative fc value, and/or a higher expression of at least one polynucleotide defined by at least one ofthe numbers 1 to 50 of Table 2.52 having a positive fc value, is indicative for the presence AML_inv(16) when AML_inv(16) distinguished from AMLJkomplext, and/or wherein a lower expression of at least one polynucleotide defined by at least one ofthe numbers 1 to 50 of Table 2.53 having a negative fc value, and/or a higher expression of at least one polynucleotide defined by at least one ofthe numbers 1 to 50 of Table 2.53 having a positive fc value, is indicative for the presence AML_inv(l 6) when AML_inv(l 6) distinguished from AML_t(15;17), and/or wherein a lower expression of at least one polynucleotide defined by at least one ofthe numbers 1 to 50 of Table 2.54 having a negative fc value, and/or a higher expression of at least one polynucleotide defined by at least one ofthe numbers 1 to 50 of Table 2.54 having a positive fc value, is indicative for the presence AML_inv(16) when AML_inv(16) distinguished from AML_t(8;21), and/or wherein a lower expression of at least one polynucleotide defined by at least one ofthe numbers 1 to 50 of Table 2.55 having a negative fc value, and/or a higher expression of at least one polynucleotide defined by at least one ofthe numbers 1 to 50 of Table 2.55 having a positive fc value, is indicative for the presence AML_inv(16) when AML_inv(16) distinguished from CLL, and or wherein a lower expression of at least one polynucleotide defined by at least one ofthe numbers 1 to 50 of Table 2.56 having a negative fc value, and/or a higher expression of at least one polynucleotide defined by at least one ofthe numbers 1 to 50 of Table 2.56 having a positive fc value, is indicative for the presence AML_inv(16) when AML_inv(16) distinguished from CML, and/or wherein a lower expression of at least one polynucleotide defined by at least one ofthe numbers 1 to 50 of Table 2.57 having a negative fc value, and/or a higher expression of at least one polynucleotide defined by at least one ofthe numbers 1 to 50 of Table 2.57 having a positive fc value, is indicative for the presence AML_inv(16) when AML_inv(16) distinguished from normal-BM, and or wherein a lower expression of at least one polynucleotide defined by at least one ofthe numbers 1 to 50 of Table 2.58 having a negative fc value, and/or a higher expression of at least one polynucleotide defined by at least one ofthe numbers 1 to 50 of Table 2.58 having a positive fc value, is indicative for the presence AML_inv(3) when AML_inv(3) distinguished from AML_komplext, and/or wherein a lower expression of at least one polynucleotide defined by at least one ofthe numbers 1 to 50 of Table 2.59 having a negative fc value, and/or a higher expression of at least one polynucleotide defined by at least one ofthe numbers 1 to 50 of Table 2.59 having a positive fc value, is indicative for the presence AML_inv(3) when AML_inv(3) distinguished from AML_t( 15 ; 17), and/or wherein a lower expression of at least one polynucleotide defined by at least one ofthe numbers 1 to 50 of Table 2.60 having a negative fc value, and/or a higher expression of at least one polynucleotide defined by at least one ofthe numbers 1 to 50 of Table 2.60 having a positive fc value, is indicative for the presence AML_inv(3) when AML_inv(3) distinguished from AML_t(8 ;21 ), and or wherein a lower expression of at least one polynucleotide defined by at least one ofthe numbers 1 to 50 of Table 2.61 having a negative fc value, and/or a higher expression of at least one polynucleotide defined by at least one ofthe numbers 1 to 50 of Table 2.61 having a positive fc value, is indicative for the presence AML_inv(3) when AML_inv(3) distinguished from CLL, and/or wherein a lower expression of at least one polynucleotide defined by at least one ofthe numbers 1 to 50 of Table 2.62 having a negative fc value, and/or a higher expression of at least one polynucleotide defined by at least one ofthe numbers 1 to 50 of Table 2.62 having a positive fc value, is indicative for the presence AML_inv(3) when AML_inv(3) distinguished from CML, and/or wherein a lower expression of at least one polynucleotide defined by at least one ofthe numbers 1 to 50 of Table 2.63 having a negative fc value, and/or a higher expression of at least one polynucleotide defined by at least one ofthe numbers 1 to 50 of Table 2.63 having a positive fc value, is indicative for the presence AML_inv(3) when AML_inv(3) distinguished from normal-BM, and/or wherein a lower expression of at least one polynucleotide defined by at least one ofthe numbers 1 to 50 of Table 2.64 having a negative fc value, and/or a higher expression of at least one polynucleotide defined by at least one ofthe numbers 1 to 50 of Table 2.64 having a positive fc value, is indicative for the presence AMLJkomplext when AML_komplext distinguished from AML_t(l 5; 17), and/or wherein a lower expression of at least one polynucleotide defined by at least one ofthe numbers 1 to 50 of Table 2.65 having a negative fc value, and/or a higher expression of at least one polynucleotide defined by at least one ofthe numbers 1 to 50 of Table 2.65 having a positive fc value, is indicative for the presence AML_komplext when AML_komplext distinguished from AML_t(8;21), and/or wherein a lower expression of at least one polynucleotide defined by at least one ofthe numbers 1 to 50 of Table 2.66 having a negative fc value, and/or a higher expression of at least one polynucleotide defined by at least one ofthe numbers 1 to 50 of Table 2.66 having a positive fc value, is indicative for the presence AMLJkomplext when AMLJ omplext distinguished from CLL, and/or wherein a lower expression of at least one polynucleotide defined by at least one ofthe numbers 1 to 50 of Table 2.67 having a negative fc value, and/or a higher expression of at least one polynucleotide defined by at least one ofthe numbers 1 to 50 of Table 2.67 having a positive fc value, is indicative for the presence AMLJcomplext when AMLJkomplext distinguished from CML, and/or wherein a lower expression of at least one polynucleotide defined by at least one ofthe numbers 1 to 50 of Table 2.68 having a negative fc value, and/or a higher expression of at least one polynucleotide defined by at least one ofthe numbers 1 to 50 of Table 2.68 having a positive fc value, is indicative for the presence AMLJcomplext when AMLJcomplext distinguished from normal-BM, and/or wherein a lower expression of at least one polynucleotide defined by at least one ofthe numbers 1 to 50 of Table 2.69 having a negative fc value, and or a higher expression of at least one polynucleotide defined by at least one ofthe numbers 1 to 50 of Table 2.69 having a positive fc value, is indicative for the presence AML_t(15;17) when AML_t(15;17) is distinguished from AML_t(8;21), and/or wherein a lower expression of at least one polynucleotide defined by at least one ofthe numbers 1 to 50 of Table 2.70 having a negative fc value, and/or a higher expression of at least one polynucleotide defined by at least one ofthe numbers 1 to 50 of Table 2.70 having a positive fc value, is indicative for the presence AML_t(15;17) when AML_t(15;17) is distinguished from CLL, and/or wherein a lower expression of at least one polynucleotide defined by at least one ofthe numbers 1 to 50 of Table 2.71 having a negative fc value, and/or a higher expression of at least one polynucleotide defined by at least one ofthe numbers 1 to 50 of Table 2.71 having a positive fc value, is indicative for the presence AML_t( 15 ; 17) when AML_t( 15 ; 17) is distinguished from CML, and/or wherein a lower expression of at least one polynucleotide defined by at least one ofthe numbers 1 to 50 of Table 2.72 having a negative fc value, and/or a higher expression of at least one polynucleotide defined by at least one ofthe numbers 1 to 50 of Table 2.72 having a positive fc value, is indicative for the presence AML_t(15;17) when AML_t(15;17) is distinguished from normal-BM, and/or wherein a lower expression of at least one polynucleotide defined by at least one ofthe numbers 1 to 50 of Table 2.73 having a negative fc value, and/or a higher expression of at least one polynucleotide defined by at least one ofthe numbers 1 to 50 of Table 2.73 having a positive fc value, is indicative for the presence AML_t(8;21) when AML_t(8;21) is distinguished from CLL, and/or wherein a lower expression of at least one polynucleotide defined by at least one ofthe numbers 1 to 50 of Table 2.74 having a negative fc value, and/or a higher expression of at least one polynucleotide defined by at least one ofthe numbers 1 to 50 of Table 2.74 having a positive fc value, is indicative for the presence AML_t(8;21) when AML_t(8;21) is distinguished from CML, and/or wherein a lower expression of at least one polynucleotide defined by at least one ofthe numbers 1 to 50 of Table 2.75 having a negative fc value, and/or a higher expression of at least one polynucleotide defined by at least one ofthe numbers 1 to 50 of Table 2.75 having a positive fc value, is indicative for the presence AML_t(8;21) when AML_t(8;21) is distinguished from normal-BM, and/or wherein a lower expression of at least one polynucleotide defined by at least one ofthe numbers 1 to 50 of Table 2.76 having a negative fc value, and/or a higher expression of at least one polynucleotide defined by at least one ofthe numbers 1 to 50 of Table 2.76 having a positive fc value, is indicative for the presence CLL when CLL is distinguished from CML, and/or wherein a lower expression of at least one polynucleotide defined by at least one ofthe numbers 1 to 50 of Table 2.77 having a negative fc value, and/or a higher expression of at least one polynucleotide defined by at least one ofthe numbers 1 to 50 of Table 2.77 having a positive fc value, is indicative for the presence CLL when CLL is distinguished from normal-BM, and/or wherein a lower expression of at least one polynucleotide defined by at least one ofthe numbers 1 to 50 of Table 2.78 having a negative fc value, and/or a higher expression of at least one polynucleotide defined by at least one ofthe numbers 1 to 50 of Table 2.78 having a positive fc value, is indicative for the presence CML when CML is distinguished from normal-BM. As used herein, the following definitions apply to the above abbreviations: t(15;17): AML with t(15;17) translocation t(8;21): AML with t(8;21) translocation inv(16): AML with inversion 16 inv(3): AML with inversion 3
CA: AML with complex aberrant karyotype
AML-MLL: AML with mutations on the mixed lineage leukaemia (MLL) gene normal karyotype (NK): AML with normal karyotype trisomy 8: AML with trisomy of chromosome 8 trisomy 11 : AML with trisomy of chromosome 11 trisomy 13: AML with trisomy of chromosome 13 monosomy 7: AML with monosomy of chromosome 7 del(5q): AML with 5q deletion del(9q): AML with 9q deletion t(6;9): AML with t(6;9) translocation del(20q): AML with 20 q deletion del(12p): AML with deletion 12 p deletion trisomy 4: AML with trisomy 4
ALL-MLL: acute lymphoblastic leukaemia with mutations on the mixed lineage leukemia (MLL) gene
ALL-Ph+: acute lymphoblastic leukaemia with genetic aberration on the Philadelphia chromosome
ALL-t(8;14): acute lymphoblastic leukemia with translocation t(8;21)
T-ALL: T cell acute lymphoblastic leukemia other B-lineage (OBL):
CML: chronic myeloid leukemia normal-BM: bone marrow from healthy volunteers
CLL: chronic lymphatic leukemia As used herein, "all other subtypes" refer to the subtypes of the present invention, i.e. to all other subtypes except for the one being under investigation
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 ofthe 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-antibody- 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 incoφorated 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 incoφorated. 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 counteφart 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 corresponds 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 incoφorated 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 leukemia 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 microtiteφlates. 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 immunoassay.
For reliably distinguishing Leukemia subtypes t(15;17), t(8;21), inv(16), inv(3), complex aberrant karyotype (CA), AML-MLL, normal karyotype (NK), AML- other, i.e. trisomy 8, trisomy 11, trisomy 13, monosomy 7, del(5q), del(9q), t(6;9); del(20q) and del(12p) and trisomy 4, ALL-MLL, ALL-Ph+, ALL-t(8;14), T-ALL, other B-lineage (OBL), CML, normal-BM, and/or CLL it is useful that the expression of more than one of the above defined markers. As a criterion for the choice of markers, the statistical significance of markers as expressed in q or p values based on the concept ofthe 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 ofthe 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 Tables
1.1-2.78 having a q- value of less than 3E-06, 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 50 of at least one ofthe Tables ofthe 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 ofthe Tables are measured.
The level of the expression of the „marker", i.e. the expression of the polynucleotide is indicative of the leukemia 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 ofthe 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 preferred 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 leukemia.
For the method of the present invention it is preferred 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 ofthe 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 ofthe 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 ofthe 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 ofthe 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.
Preferred 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 resoance energy transfer between two adjacenly hybridized probes as used in the LightCycler® format described in U.S.
6,174,670.
A preferred 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 incoφorated during the transcription reaction. The purified cRNA is applied to commercially available arrays which can be obtained e.g. from Affymetrix. The hybridized cRNA is detected according to the methods described in Example 3. The arrays 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 0 373 203, 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 preferred embodiment, the expression level ofthe polynucleotides or polypeptides is detected using a compound which specifically binds to the polynucleotide ofthe polypeptide ofthe 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 preferred 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 Kδhler 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 immuno 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 ofthe labelling compounds and methods described supra. In another preferred embodiment of the present invention, the method for distinguishing leukemia subtypes is carried out on an array.
In general, an "array" or "microarray" refers to a linear or two- or three dimensional arrangement 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 "array". As known to the person skilled in the art, a microarray usually refers to a miniaturised array arrangement, with the probes being attached to a density of at least about 10, 20, 50, 100 nucleic acid molecules referring to different or the same genes per cm2. Furthermore, where appropriate an array can be referred to as "gene chip". The array 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 array fabrication is well-known to the person skilled in the art. In the following, the process for preparing a nucleic acid array 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 information 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 array. The liquid containing the amplified probes can be deposited on the array 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 array.
In a preferred embodiment, the array is a high density oligonucleotide (oligo) array using a light-directed chemical synthesis process, employing the so-called photolithography technology. Unlike common cDNA arrays, oligo arrays
(according to the Affymetrix technology) use a single-dye technology. Given the sequence information ofthe markers, the sequence can be synthesized directly onto the array, thus, bypassing the need for physical intermediates, such as PCR products, required for making cDNA arrays. For this puφose, 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 carried out in a robotics system including robotic plating and a robotic liquid transfer system, e.g. using microfluidics, i.e. channelled structured.
A particular preferred method according to the present invention is as follows:
1. Obtaining a sample, e.g. bone marrow or peripheral blood aliquots, from a patient having leukemia
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 microarrays
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, and/or 2 for the manufacturing of a diagnostic for distinguishing Leukemia subtypes. The use of the present invention is particularly advantageous for distinguishing leukemia subtypes in an individual having leukemia. The use of said markers for diagnosis of leukemia subtypes, preferably based on microarray 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 cytomoφhology 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 2 for distinguishing leukemia subtypes, in combination with suitable auxiliaries. Suitable auxiliaries, as used herein, include buffers, enzymes, labelling compounds, and the like. In a preferred embodiment, the marker contained in the kit is a nucleic acid molecule which is capable of hybridizing to the mRNA corresponding 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 preferred embodiment, the diagnostic kit contains at least one reference for a t(15;17), t(8;21), inv(16), inv(3), complex aberrant karyotype (CA), AML- MLL, normal karyotype (NK), AML-other, i.e. trisomy 8, trisomy 11, trisomy 13, monosomy 7, del(5q), del(9q), t(6;9); del(20q) and del(12p) and trisomy 4, ALL-
MLL, ALL-Ph+, ALL-t(8;14), T-ALL, other B-lineage (OBL), CML, normal-BM, and/or CLL leukemia 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 leukemia subtypes t(15;17), t(8;21), inv(16), inv(3), complex aberrant karyotype (CA), AML-MLL, normal karyotype (NK), AML-other, i.e. trisomy 8, trisomy 11, trisomy 13, monosomy 7, del(5q), del(9q), t(6;9); del(20q) and del(12p) and trisomy 4, ALL-MLL, ALL-Ph+, ALL-t(8;14), T-ALL, other B- lineage (OBL), CML, normal-BM, and/or CLL 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 2, 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 corresponding to a certain expression level which are obtained by the microarray 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 microarray 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 referred 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 microarray 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 correct 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 ofthe 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 preferred 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 ofthe present invention, e.g. like an interchangeable module, in order to use another data memory chip containing a different reference data bank.
The apparatus ofthe 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 microarray 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 ofthe reference data bank obtainable by the above method. For this puφose, 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 ofthe 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 incoφorated 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 microarray containing the hybridized nucleic acids.
In another embodiment, the present invention refers to a reference data bank for extinguishing leukemia subtypes t(15;17), t(8;21), inv(16), inv(3), complex aberrant karyotype (CA), AML-MLL, normal karyotype (NK), AML-other, i.e. trisomy 8, trisomy 11, trisomy 13, monosomy 7, del(5q), del(9q), t(6;9); del(20q) and del(12p) and trisomy 4, ALL-MLL, ALL-Ph+, ALL-t(8;14), T-ALL, other B- lineage (OBL), CML, normal-BM, and/or CLL 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 7, 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 ofthe invention:
TABLES 1.1 to 2.78 Tables 1.1 to 2.78 show leukemia subtype analysis of t(15;17), t(8;21), inv(16), inv(3), complex aberrant karyotype (CA), AML-MLL, normal karyotype (NK), AML-other, i.e. trisomy 8, trisomy 11, trisomy 13, monosomy 7, del(5q), del(9q), t(6;9); del(20q) and del(12p) and trisomy 4, ALL-MLL, ALL-Ph+, ALL-t(8;14), T- ALL, other B-lineage (OBL), CML, normal-BM, and/or CLL. 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 2.78 are accompanied with explanatory tables (Table 1.1 A to 2.78A) 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 So far comprehensive diagnosis of leukemia requires a combination of cytomoφhology, immunophenotyping, and genetic methods. We aimed at developing a diagnostic tool based only on gene expression profiling to accurately predict all clinically relevant subtypes of leukemia. Therefore, we analyzed samples from 540 patients at diagnosis using oligonucleotide microarrays representing 33,000 different genes (U133 set, Affymetrix). The following leukemia subtypes were included in this study: 367 AML (20 1(15;17); 25 t(8;21); 25 inv(16); 18 inv(3); 34 complex aberrant karyotype (CA); 30 AML-MLL; 158 normal karyotype (NK); 57 AML-other, i.e. trisomy 8 (n=12), trisomy 11 (n=7), trisomy 13 (n=7), monosomy 7 (n=9), del(5q) (n=7), del(9q) (n=9), t(6;9) (n=3); del(20q) and del(12p) and trisomy 4 one case each); 85 ALL (17 ALL-MLL; 21
ALL-Ph+; 12 ALL-t(8;14); 23 T-ALL; 12 other B-lineage (OBL)), 46 CML, 34 CLL, and 8 bone marrows from healthy volunteers (n-BM). To identify differentially expressed genes we applied ANOVA and t-test-statistics (Welch t- test). To assess the false discovery rate we calculated q values according to Storey et al. (PNAS, 2003). To estimate diagnostic accuracy based on gene expression signatures, we designated a training set consisting of 2/3 of cases and a test set with 1/3 of cases. Assignment of cases to training and test set was randomized and balanced by diagnosis. Based on the fraining set we built Support Vector Machine (SVM) models. Classification accuracy was assessed in the independent test set. In the first analysis five main categories AML, ALL, CML, CLL, and n-BM were distinguished in the test set with an accuracy of 96% (177/184 correctly assigned). In a second analysis the following 13 subtypes were included: ALL-MLL, ALL-
Ph+, T-ALL, ALL-t(8;14), AML-t(8;21), AML-inv(16), AML-t(15;17), AML- MLL, AML-inv(3), AML-CA, AML-NK, CLL, and CML. 151/154 cases of the test set were correctly assigned (98%). Only two cases with AML-CA and one case with AML-NK were misclassified. In a third analysis n-BM, AML-other and ALL- OBL were added to the 13 subtypes. The accuracy was reduced to 88% (159/180).
Categories with 100% sensitivity and specificity each were: n-BM, CLL, CML, ALL-MLL, ALL-t(8;14), AML-t(15;17), and AML-inv(16). AML-other and ALL- OBL, respectively, are considered genetically heterogeneous diseases and are not characterized by a specific gene expression profile. This may have caused the reduced accuracy in the latter SVM analysis. In conclusion, we were able to identify distinct expression profiles for all clinically and prognostically relevant leukemia subtypes based on gene expression data. Sensitivity and specificity were very high when specific leukemia subtypes were included into the analysis. Even the subgroup AML-NK was predicted with high accuracy. Using gene expression profiling as a robust diagnostic tool to correctly subclassify leukemias is a realistic goal and may guide relevant therapeutic consequences in the near future.
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 microarray probesets.
Affymetrix Probeset Annotation
All annotation data of GeneChip® arrays are extracted from the NetAffic™ Analysis Center (internet website: www.affymetrix.com). Files for U133 set arrays, including U133A and U133B microarrays 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;31(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. Microarray probesets, for example found to be differentially expressed between different types of leukemia samples are further described by additional information.
The fields are ofthe following types:
1. GeneChip Array Information
2. Probe Design Information 3. Public Domain and Genomic References
1. GeneChip Array Information
HG-U133 ProbeSet D: HG-U133 ProbeSet_ID describes the probe set identifier. Examples are:
200007_at, 20001 l_s_at, 200012_x_at.
GeneChip:
The description ofthe GeneChip probe array name where the respective probeset is represented. Examples are: Affymetrix Human Genome U133A Array or
Affymetrix Human Genome U133B Array.
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 ofthe data in this section come from LocusLink and UniGene databases, and are annotations ofthe 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:
Microarray 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 IO7 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 incoφorated during the following in vitro transcription reaction (Enzo BioArray HighYield
RNA Transcript Labeling Kit, Enzo Diagnostics). After quantification by spectrophotometric measurements and 260/280 absorbance values assessment for quality control ofthe 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 microarrays (300 μl final volume). Washing and staining of the probe arrays was performed according to the recommended Fluidics Station protocol (EukGE-WS2v4). Affymetrix Microarray Suite software (version 5.0.1) extracted fluorescence signal intensities from each feature on the microarrays as detected by confocal laser scanning according to the manufacturer's recommendations. Expression analysis quality assessment parameters included visiual array inspection ofthe scanned image for the presence of image artifacts and correct 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 ofthe 3' probe sets for GAPDH are compared to the Signal values of the corresponding 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 Microarray Analysis Suite MAS 5.0 software package.
Method 2:
Bone marrow (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 microarray analysis the GeneChip System (Affymetrix, Santa Clara, CA, USA) is used. The targets for GeneChip analysis are prepared according to the current Expression Analysis. Briefly, frozen lysates ofthe 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 incoφorated during the in vitro transcription reaction (Enzo® Bio Array™
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 microarrays. Before expression profiling
Test3 Probe Arrays (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 arrays (Affymetrix). Washing and staining the Probe arrays is performed as described (siehe Affymetrix-Original- Literatur (LOCKHART und LIPSHUTZ). The Affymetrix software (Microarray Suite, Version 4.0.1) extracted fluorescence intensities from each element on the arrays as detected by confocal laser scanning according to the manufacturers recommendations.
Table 1
1. One-Versus-AII (OVA)
Figure imgf000049_0001
Table 1
Figure imgf000050_0001
Table 1
Figure imgf000051_0001
Table 1
Figure imgf000052_0001
Table 1
Figure imgf000053_0001
Table 1
Figure imgf000054_0001
54 I aoie
Figure imgf000055_0001
Table 1
Figure imgf000056_0001
56 Table 1
Figure imgf000057_0001
57 Table 1
Figure imgf000058_0001
58 Table 1
Figure imgf000059_0001
Figure imgf000060_0001
Figure imgf000061_0001
61 Table 1
Figure imgf000062_0001
62 Table 1
Figure imgf000063_0001
63 Table 1
Figure imgf000064_0001
Table 2.1-2.78
Table 2
2. All-Pairs (AP)
Figure imgf000065_0001
Table 2.1-2.78
Figure imgf000066_0001
Table 2.1-2.78
Figure imgf000067_0001
Table 2.1-2.78
Figure imgf000068_0001
Table 2.1-2.78
Figure imgf000069_0001
Table 2.1-2.78
Figure imgf000070_0001
Table 2.1-2.78
Figure imgf000071_0001
Table 2.1-2.78 71
Figure imgf000072_0001
Table 2.1-2.78
Figure imgf000073_0001
Table 2.1-2.78
Figure imgf000074_0001
Table^.1-2.78
Figure imgf000075_0001
Table 2.1-2.78
Figure imgf000076_0001
Table 2.1-2.78
Figure imgf000077_0001
Table 2.1-2.78
Figure imgf000078_0001
Table 2.1-2.78
Figure imgf000079_0001
Table 2.1-2.78
Figure imgf000080_0001
Table 2.1-2.78
Figure imgf000081_0001
Table 2.1-2.78
Figure imgf000082_0001
Table 2.1-2.78
Figure imgf000083_0001
Table 2.1-2.78
Figure imgf000084_0001
Table 2.1-2.78
Figure imgf000085_0001
Table 2.1-2.78
Figure imgf000086_0001
Table 2.1-2.78
Figure imgf000087_0001
Table 2.1-2.78
Figure imgf000088_0001
Table 2.1-2.78
Figure imgf000089_0001
Table 2.1-2.78
Figure imgf000090_0001
Table 2.1-2.78
Figure imgf000091_0001
Table 2.1-2.78
Figure imgf000092_0001
Table 2.1-2.78
Figure imgf000093_0001
Table 2.1-2:78
Figure imgf000094_0001
Table 2.1-2.78
Figure imgf000095_0001
Table 2.1-2.78
Figure imgf000096_0001
Table 2.1-2.78
Figure imgf000097_0001
Table 2.1-2.78
Figure imgf000098_0001
Table 2.1-2.78
Figure imgf000099_0001
Table 2.1-2.78
Figure imgf000100_0001
Table 2.1-2.78
Figure imgf000101_0001
Table 2.1-2.78
Figure imgf000102_0001
Table 2.1-2.78
Figure imgf000103_0001
Table 2.1-2.78
Figure imgf000104_0001
Table 2.1-2.78
Figure imgf000105_0001
Table 2.1-2.78
Figure imgf000106_0001
Table 2.1-2.78
Figure imgf000107_0001
Table 2.1-2.78
Figure imgf000108_0001
Table 2.1-2.78
Figure imgf000109_0001
Table 2.1-2.78
Figure imgf000110_0001
Table 2.1-2.78
Figure imgf000111_0001
Table 2.1-2.78
Figure imgf000112_0001
Table 2.1-2.78
Figure imgf000113_0001
Table 2.1-2.78
Figure imgf000114_0001
Table 2.1-2.78
Figure imgf000115_0001
Table 2.1-2.78
Figure imgf000116_0001
Table 2.1-2.78
Figure imgf000117_0001
Table 2.1-2.78
Figure imgf000118_0001
Table 2.1-2.78
Figure imgf000119_0001
Table 2.1-2.78
Figure imgf000120_0001
Tabfe 2.1-2.78
Figure imgf000121_0001
Table 2.1-2.78
Figure imgf000122_0001
Table 2.1-2.78
Figure imgf000123_0001
Table 2.1-2.78
Table 2.1-2.78
Figure imgf000125_0001
Table 2.1=2.78
Figure imgf000126_0001
Table 2.1-2.78
Figure imgf000127_0001
Table 2.1-2.78
Figure imgf000128_0001
Table 2.1-2.78
Figure imgf000129_0001
Table 2.1-2.78
Figure imgf000130_0001
Table 2.1-2.78
Figure imgf000131_0001
Table 2.1-2.78
Figure imgf000132_0001
Table 2.1-2.78
Figure imgf000133_0001
Table 2.1-2.78
Figure imgf000134_0001
Table 2.1-2.78
Figure imgf000135_0001
Table 2.1-2.78
Figure imgf000136_0001
Table 2.1-2.78
Figure imgf000137_0001
Table 2.1-2.78
Figure imgf000138_0001
Table 2.1-2.78
Figure imgf000139_0001
Table 2.1-2.78
Figure imgf000140_0001
Table 2.1-2.78
Figure imgf000141_0001
Table 2.1-2.78
Figure imgf000142_0001
Table 2.1-2.78
Figure imgf000143_0001
Table 2.1-2.78
Figure imgf000144_0001
Table 2.1-2.78
Figure imgf000145_0001
Table 2.1-2.78
Figure imgf000146_0001
Table 2.1-2.78
Figure imgf000147_0001
Table 2.1-2.78
Figure imgf000148_0001
Table 2.1-2.78
Figure imgf000149_0001
Table 2.1-2.78
Figure imgf000150_0001
Table 2.1-2.78
Figure imgf000151_0001
Table 2.1-2.78
Figure imgf000152_0001
Table 2.1-2.78
Figure imgf000153_0001
Table 2.1-2.78
Figure imgf000154_0001
Table 2.1-2.78
Figure imgf000155_0001
Table 2.1-2.78
Figure imgf000156_0001
Table 2.1-2.78
Figure imgf000157_0001
Table 2.1-2.78
Figure imgf000158_0001
Table 2.1-2.78
Figure imgf000159_0001

Claims

Claims
1. A method for distinguishing leukemia subtypes t(15;17), t(8;21), inv(16), inv(3), complex aberrant karyotype (CA), AML-MLL, normal karyotype (NK), AML-other (trisomy 8, trisomy 11, trisomy 13, monosomy 7, del(5q), del(9q), t(6;9); del(20q) and del(12p) and trisomy 4), ALL-MLL, ALL-Ph+, ALL-t(8;14), T-ALL, other B-lineage (OBL), CML, normal-BM, and or CLL 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 and/or 2, wherein a lower expression of at least one polynucleotide defined by at least one ofthe numbers 1 to 50 of Table 1.1 having a negative fc value, and/or a higher expression of at least one polynucleotide defined by at least one ofthe numbers 1 to 50 of Table 1.1 having a positive fc value, is indicative for the presence ALLJVILL when ALLJVILL is distinguished from all other subtypes, and/or wherein a lower expression of at least one polynucleotide defined by at least one ofthe numbers 1 to 50 of Table 1.2 having a negative fc value, and or a higher expression of at least one polynucleotide defined by at least one ofthe numbers 1 to 50 of Table 1.2 having a positive fc value, is indicative for the presence ALLJPh÷ when ALL_Ph+ is distinguished from all other subtypes, and/or wherein a lower expression of at least one polynucleotide defined by at least one ofthe numbers 1 to 50 of Table 1.3 having a negative fc value, and/or a higher expression of at least one polynucleotide defined by at least one ofthe numbers 1 to 50 of Table 1.3 having a positive fc value, is indicative for the presence ALL_T lineage when ALL_T lineage is distinguished from all other subtypes, and or wherein a lower expression of at least one polynucleotide defined by at least one ofthe numbers 1 to 50 of Table 1.4 having a negative fc value, and/or a higher expression of at least one polynucleotide defined by at least one ofthe numbers 1 to 50 of Table 1.4 having a positive fc value, is indicative for the presence ALL_t(8;14) when ALL_t(8; 14) is distinguished from all other subtypes, and/or wherein a lower expression of at least one polynucleotide defined by at least one ofthe numbers 1 to 50 of Table 1.5 having a negative fc value, and/or a higher expression of at least one polynucleotide defined by at least one ofthe numbers 1 to 50 of Table 1.5 having a positive fc value, is indicative for the presence AMLJVILL when AMLJVILL is distinguished from all other subtypes, and/or wherein a lower expression of at least one polynucleotide defined by at least one ofthe numbers 1 to 50 of Table 1.6 having a negative fc value, and/or a higher expression of at least one polynucleotide defined by at least one ofthe numbers 1 to 50 of Table 1.6 having a positive fc value, is indicative for the presence AML_inv(16) when AML_inv(16) is distinguished from all other subtypes, and/or wherein a lower expression of at least one polynucleotide defined by at least one ofthe numbers 1 to 50 of Table 1.7 having a negative fc value, and/or a higher expression of at least one polynucleotide defined by at least one ofthe numbers 1 to 50 of Table 1.7 having a positive fc value, is indicative for the presence AML_inv(3) when AML_inv(3) is distinguished from all other subtypes, and/or wherein a lower expression of at least one polynucleotide defined by at least one ofthe numbers 1 to 50 of Table 1.8 having a negative fc value, and/or a higher expression of at least one polynucleotide defined by at least one ofthe numbers 1 to 50 of Table 1.8 having a positive fc value, is indicative for the presence AMLJcomplext when AMLJcomplext is distinguished from all other subtypes, and/or wherein a lower expression of at least one polynucleotide defined by at least one ofthe numbers 1 to 50 of Table 1.9 having a negative fc value, and/or a higher expression of at least one polynucleotide defined by at least one ofthe numbers 1 to 50 of Table 1.9 having a positive fc value, is indicative for the presence AML _t( 15 ; 17) when AML_t( 15 ; 17) is distinguished from all other subtypes, and/or wherein a lower expression of at least one polynucleotide defined by at least one ofthe numbers 1 to 50 of Table 1.10 having a negative fc value, and/or a higher expression of at least one polynucleotide defined by at least one ofthe numbers 1 to 50 of Table 1.10 having a positive fc value, is indicative for the presence AML_t(8;21) when AML_t(8;21) is distinguished from all other subtypes, and/or wherein a lower expression of at least one polynucleotide defined by at least one ofthe numbers 1 to 50 of Table 1.11 having a negative fc value, and/or a higher expression of at least one polynucleotide defined by at least one ofthe numbers 1 to 50 of Table 1.11 having a positive fc value, is indicative for the presence CLL when CLL is distinguished from all other subtypes, and/or wherein a lower expression of at least one polynucleotide defined by at least one ofthe numbers 1 to 50 of Table 1.12 having a negative fc value, and/or a higher expression of at least one polynucleotide defined by at least one ofthe numbers 1 to 50 of Table 1.12 having a positive fc value, is indicative for the presence CML when CML is distinguished from all other subtypes, and/or wherein a lower expression of at least one polynucleotide defined by at least one ofthe numbers 1 to 50 of Table 1.13 having a negative fc value, and/or a higher expression of at least one polynucleotide defined by at least one ofthe numbers 1 to 50 of Table 1.13 having a positive fc value, is indicative for the presence normal-BM when normal-BM is distinguished from all leukemia subtypes, and/or wherein a lower expression of at least one polynucleotide defined by at least one ofthe numbers 1 to 50 of Table 2.1 having a negative fc value, and or a higher expression of at least one polynucleotide defined by at least one ofthe numbers 1 to 50 of Table 2.1 having a positive fc value, is indicative for the presence ALLJVILL when ALLJVILL is distinguished from ALLJPh+, and/or wherein a lower expression of at least one polynucleotide defined by at least one ofthe numbers 1 to 50 of Table 2.2 having a negative fc value, and/or a higher expression of at least one polynucleotide defined by at least one ofthe numbers 1 to 50 of Table 2.2 having a positive fc value, is indicative for the presence ALLJVILL when ALLJVILL is distinguished from ALL_T lineage, and/or wherein a lower expression of at least one polynucleotide defined by at least one ofthe numbers 1 to 50 of Table 2.3 having a negative fc value, and/or a higher expression of at least one polynucleotide defined by at least one ofthe numbers 1 to 50 of Table 2.3 having a positive fc value, is indicative for the presence ALLJVILL when ALLJMLL is distinguished from ALLJ(8; 14), and/or wherein a lower expression of at least one polynucleotide defined by at least one ofthe numbers 1 to 50 of Table 2.4 having a negative fc value, and/or a higher expression of at least one polynucleotide defined by at least one ofthe numbers 1 to 50 of Table 2.4 having a positive fc value, is indicative for the presence ALLJVILL when ALLJVILL is distinguished from AMLJVILL, and/or wherein a lower expression of at least one polynucleotide defined by at least one ofthe numbers 1 to 50 of Table 2.5 having a negative fc value, and/or a higher expression of at least one polynucleotide defined by at least one ofthe numbers 1 to 50 of Table 2.5 having a positive fc value, is indicative for the presence ALLJVILL when ALLJVILL is distinguished from AML_inv(16), and/or wherein a lower expression of at least one polynucleotide defined by at least one ofthe numbers 1 to 50 of Table 2.6 having a negative fc value, and/or a higher expression of at least one polynucleotide defined by at least one ofthe numbers 1 to 50 of Table 2.6 having a positive fc value, is indicative for the presence ALLJVILL when ALLJVILL is distinguished from AML_inv(3), and/or wherein a lower expression of at least one polynucleotide defined by at least one ofthe numbers 1 to 50 of Table 2.7 having a negative fc value, and/or a higher expression of at least one polynucleotide defined by at least one ofthe numbers 1 to 50 of Table 2.7 having a positive fc value, is indicative for the presence ALLJVILL when ALLJVILL is distinguished from AMLJcomplext, and/or wherein a lower expression of at least one polynucleotide defined by at least one ofthe numbers 1 to 50 of Table 2.8 having a negative fc value, and/or a higher expression of at least one polynucleotide defined by at least one ofthe numbers 1 to 50 of Table 2.8 having a positive fc value, is indicative for the presence ALLJVILL when ALLJVILL is distinguished from AML_t(15;17), and/or wherein a lower expression of at least one polynucleotide defined by at least one ofthe numbers 1 to 50 of Table 2.9 having a negative fc value, and/or a higher expression of at least one polynucleotide defined by at least one ofthe numbers 1 to 50 of Table 2.9 having a positive fc value, is indicative for the presence ALLJVILL when ALLJVILL is distinguished from AML_t(8;21), and/or wherein a lower expression of at least one polynucleotide defined by at least one ofthe numbers 1 to 50 of Table 2.10 having a negative fc value, and/or a higher expression of at least one polynucleotide defined by at least one ofthe numbers 1 to 50 of Table 2.10 having a positive fc value, is indicative for the presence ALLJVILL when ALLJVILL is distinguished from CLL, and/or wherein a lower expression of at least one polynucleotide defined by at least one ofthe numbers 1 to 50 of Table 2.11 having a negative fc value, and/or a higher expression of at least one polynucleotide defined by at least one ofthe numbers 1 to 50 of Table 2.11 having a positive fc value, is indicative for the presence ALLJVILL when ALLJMLL is distinguished from CML, and/or wherein a lower expression of at least one polynucleotide defined by at least one ofthe numbers 1 to 50 of Table 2.12 having a negative fc value, and/or a higher expression of at least one polynucleotide defined by at least one ofthe numbers 1 to 50 of Table 2.12 having a positive fc value, is indicative for the presence ALLJVILL when ALLJVILL is distinguished from normal-BM, and/or wherein a lower expression of at least one polynucleotide defined by at least one ofthe numbers 1 to 50 of Table 2.13 having a negative fc value, andor a higher expression of at least one polynucleotide defined by at least one ofthe numbers 1 to 50 of Table 2.13 having a positive fc value, is indicative for the presence ALL_Ph+ when ALL_Ph+ is distinguished from ALLJT lineage, and/or wherein a lower expression of at least one polynucleotide defined by at least one ofthe numbers 1 to 50 of Table 2.14 having a negative fc value, and/or a higher expression of at least one polynucleotide defined by at least one ofthe numbers 1 to 50 of Table 2.14 having a positive fc value, is indicative for the presence ALL_Ph+ when ALL_Ph+ is distinguished from ALL_t(8;14), and/or wherein a lower expression of at least one polynucleotide defined by at least one ofthe numbers 1 to 50 of Table 2.15 having a negative fc value, and/or a higher expression of at least one polynucleotide defined by at least one ofthe numbers 1 to 50 of Table 2.15 having a positive fc value, is indicative for the presence ALL_Ph+ when ALL_Ph+ is distinguished from AMLJVILL, and/or wherein a lower expression of at least one polynucleotide defined by at least one ofthe numbers 1 to 50 of Table 2.16 having a negative fc value, and/or a higher expression of at least one polynucleotide defined by at least one ofthe numbers 1 to 50 of Table 2.16 having a positive fc value, is indicative for the presence ALL_Ph+ when ALL_Ph+ is distinguished from AML_inv( 16), and/or wherein a lower expression of at least one polynucleotide defined by at least one ofthe numbers 1 to 50 of Table 2.17 having a negative fc value, and/or a higher expression of at least one polynucleotide defined by at least one ofthe numbers 1 to 50 of Table 2.17 having a positive fc value, is indicative for the presence ALL_Ph+ when ALL_Ph+ is distinguished from AML_inv(3), and/or wherein a lower expression of at least one polynucleotide defined by at least one ofthe numbers 1 to 50 of Table 2.18 having a negative fc value, and/or a higher expression of at least one polynucleotide defined by at least one ofthe numbers 1 to 50 of Table 2.18 having a positive fc value, is indicative for the presence ALL_Ph+ when ALL_Ph+ is distinguished from AMLJcomplext, and/or wherein a lower expression of at least one polynucleotide defined by at least one ofthe numbers 1 to 50 of Table 2.19 having a negative fc value, and or a higher expression of at least one polynucleotide defined by at least one ofthe numbers 1 to 50 of Table 2.19 having a positive fc value, is indicative for the presence ALL_Ph+ when ALL_Ph+ is distinguished from AML_t( 15 ; 17), and/or wherein a lower expression of at least one polynucleotide defined by at least one ofthe numbers 1 to 50 of Table 2.20 having a negative fc value, and/or a higher expression of at least one polynucleotide defined by at least one ofthe numbers 1 to 50 of Table 2.20 having a positive fc value, is indicative for the presence ALL_Ph+ when ALL_Ph+ is distinguished from AML_t(8;21), and/or wherein a lower expression of at least one polynucleotide defined by at least one ofthe numbers 1 to 50 of Table 2.21 having a negative fc value, and/or a higher expression of at least one polynucleotide defined by at least one ofthe numbers 1 to 50 of Table 2.21 having a positive fc value, is indicative for the presence ALL_Ph+ when ALL_Ph+ is distinguished from CLL, and/or wherein a lower expression of at least one polynucleotide defined by at least one ofthe numbers 1 to 50 of Table 2.22 having a negative fc value, and/or a higher expression of at least one polynucleotide defined by at least one ofthe numbers 1 to 50 of Table 2.22 having a positive fc value, is indicative for the presence ALL_Ph+ when ALL_Ph+ is distinguished from CML, and/or wherein a lower expression of at least one polynucleotide defined by at least one ofthe numbers 1 to 50 of Table 2.23 having a negative fc value, and or a higher expression of at least one polynucleotide defined by at least one ofthe numbers 1 to 50 of Table 2.23 having a positive fc value, is indicative for the presence ALL_Ph+ when ALL_Ph+ is distinguished from normal-BM, and/or wherein a lower expression of at least one polynucleotide defined by at least one ofthe numbers 1 to 50 of Table 2.24 having a negative fc value, and/or a higher expression of at least one polynucleotide defined by at least one ofthe numbers 1 to 50 of Table 2.24 having a positive fc value, is indicative for the presence ALL_T lineage when ALL_T lineage distinguished from ALL_t(8; 14), and/or wherein a lower expression of at least one polynucleotide defined by at least one ofthe numbers 1 to 50 of Table 2.25 having a negative fc value, and/or a higher expression of at least one polynucleotide defined by at least one ofthe numbers 1 to 50 of Table 2.25 having a positive fc value, is indicative for the presence ALL_T lineage when ALLJT lineage distinguished from AML_MLL, and/or wherein a lower expression of at least one polynucleotide defined by at least one ofthe numbers 1 to 50 of Table 2.26 having a negative fc value, and/or a higher expression of at least one polynucleotide defined by at least one ofthe numbers 1 to 50 of Table 2.26 having a positive fc value, is indicative for the presence ALLJT lineage when ALLJT lineage distinguished from AML_inv(16), and/or wherein a lower expression of at least one polynucleotide defined by at least one ofthe numbers 1 to 50 of Table 2.27 having a negative fc value, and/or a higher expression of at least one polynucleotide defined by at least one ofthe numbers 1 to 50 of Table 2.27 having a positive fc value, is indicative for the presence ALLJT lineage when ALLJT lineage distinguished from AML_inv(3), and/or wherein a lower expression of at least one polynucleotide defined by at least one ofthe numbers 1 to 50 of Table 2.28 having a negative fc value, and/or a higher expression of at least one polynucleotide defined by at least one ofthe numbers 1 to 50 of Table 2.28 having a positive fc value, is indicative for the presence ALLJT lineage when ALLJT lineage distinguished from AMLJcomplext, and/or wherein a lower expression of at least one polynucleotide defined by at least one ofthe numbers 1 to 50 of Table 2.29 having a negative fc value, and/or a higher expression of at least one polynucleotide defined by at least one ofthe numbers 1 to 50 of Table 2.29 having a positive fc value, is indicative for the presence ALLJT lineage when ALLJT lineage distinguished from AMLJ(15;17), and/or wherein a lower expression of at least one polynucleotide defined by at least one ofthe numbers 1 to 50 of Table 2.30 having a negative fc value, and/or a higher expression of at least one polynucleotide defined by at least one ofthe numbers 1 to 50 of Table 2.30 having a positive fc value, is indicative for the presence ALLJT lineage when ALLJT lineage distinguished from AML_t(8;21), and/or wherein a lower expression of at least one polynucleotide defined by at least one ofthe numbers 1 to 50 of Table 2.31 having a negative fc value, and/or a higher expression of at least one polynucleotide defined by at least one ofthe numbers 1 to 50 of Table 2.31 having a positive fc value, is indicative for the presence ALLJT lineage when ALLJT lineage distinguished from CLL, and/or wherein a lower expression of at least one polynucleotide defined by at least one ofthe numbers 1 to 50 of Table 2.32 having a negative fc value, and or a higher expression of at least one polynucleotide defined by at least one ofthe numbers 1 to 50 of Table 2.32 having a positive fc value, is indicative for the presence ALLJT lineage when ALLJT lineage distinguished from CML, and/or wherein a lower expression of at least one polynucleotide defined by at least one ofthe numbers 1 to 50 of Table 2.33 having a negative fc value, and or a higher expression of at least one polynucleotide defined by at least one ofthe numbers 1 to 50 of Table 2.33 having a positive fc value, is indicative for the presence ALLJT lineage when ALLJT lineage distinguished from normal-BM, and/or wherein a lower expression of at least one polynucleotide defined by at least one ofthe numbers 1 to 50 of Table 2.34 having a negative fc value, and/or a higher expression of at least one polynucleotide defined by at least one ofthe numbers 1 to 50 of Table 2.34 having a positive fc value, is indicative for the presence ALL_t(8;14) when ALL_t(8;14) distinguished from AMLJVILL, and/or wherein a lower expression of at least one polynucleotide defined by at least one ofthe numbers 1 to 50 of Table 2.35 having a negative fc value, and/or a higher expression of at least one polynucleotide defined by at least one ofthe numbers 1 to 50 of Table 2.35 having a positive fc value, is indicative for the presence ALL_t(8 ; 14) when ALL_t(8 ; 14) distinguished from AML_inv(16), and/or wherein a lower expression of at least one polynucleotide defined by at least one ofthe numbers 1 to 50 of Table 2.36 having a negative fc value, and/or a higher expression of at least one polynucleotide defined by at least one ofthe numbers 1 to 50 of Table 2.36 having a positive fc value, is indicative for the presence ALL_t(8;14) when ALL_t(8;14) distinguished from AML_inv(3), and/or wherein a lower expression of at least one polynucleotide defined by at least one ofthe numbers 1 to 50 of Table 2.37 having a negative fc value, and/or a higher expression of at least one polynucleotide defined by at least one ofthe numbers 1 to 50 of Table 2.37 having a positive fc value, is indicative for the presence ALL_t(8;14) when ALLJ(8;14) distinguished from AMLJcomplext, and/or wherein a lower expression of at least one polynucleotide defined by at least one ofthe numbers 1 to 50 of Table 2.38 having a negative fc value, and/or a higher expression of at least one polynucleotide defined by at least one ofthe numbers 1 to 50 of Table 2.38 having a positive fc value, is indicative for the presence ALLJ(8;14) when ALL_t(8;14) distinguished from AML_t(15;17), and/or wherein a lower expression of at least one polynucleotide defined by at least one ofthe numbers 1 to 50 of Table 2.39 having a negative fc value, and/or a higher expression of at least one polynucleotide defined by at least one ofthe numbers 1 to 50 of Table 2.39 having a positive fc value, is indicative for the presence ALL_t(8;14) when ALL_t(8;14) distinguished from AML :(8;21), and/or wherein a lower expression of at least one polynucleotide defined by at least one ofthe numbers 1 to 50 of Table 2.40 having a negative fc value, and/or a higher expression of at least one polynucleotide defined by at least one ofthe numbers 1 to 50 of Table 2.40 having a positive fc value, is indicative for the presence ALL_t(8 ; 14) when ALL_t(8 ; 14) distinguished from CLL, and/or wherein a lower expression of at least one polynucleotide defined by at least one ofthe numbers 1 to 50 of Table 2.41 having a negative fc value, and/or a higher expression of at least one polynucleotide defined by at least one ofthe numbers 1 to 50 of Table 2.41 having a positive fc value, is indicative for the presence ALL_t(8;14) when ALL_t(8;14) distinguished from CML, and/or wherein a lower expression of at least one polynucleotide defined by at least one ofthe numbers 1 to 50 of Table 2.42 having a negative fc value, and/or a higher expression of at least one polynucleotide defined by at least one ofthe numbers 1 to 50 of Table 2.42 having a positive fc value, is indicative for the presence ALL_t(8;14) when ALL_t(8;14) distinguished from normal-BM, and/or wherein a lower expression of at least one polynucleotide defined by at least one ofthe numbers 1 to 50 of Table 2.43 having a negative fc value, and/or a higher expression of at least one polynucleotide defined by at least one ofthe numbers 1 to 50 of Table 2.43 having a positive fc value, is indicative for the presence AML JVILL when AML_MLL distinguished from AML_inv(16), and/or wherein a lower expression of at least one polynucleotide defined by at least one ofthe numbers 1 to 50 of Table 2.44 having a negative fc value, and/or a higher expression of at least one polynucleotide defined by at least one ofthe numbers 1 to 50 of Table 2.44 having a positive fc value, is indicative for the presence AMLJVILL when AMLJVILL distinguished from AML_inv(3), and/or wherein a lower expression of at least one polynucleotide defined by at least one ofthe numbers 1 to 50 of Table 2.45 having a negative fc value, and/or a higher expression of at least one polynucleotide defined by at least one ofthe numbers 1 to 50 of Table 2.45 having a positive fc value, is indicative for the presence AMLJVILL when AMLJVILL distinguished from AMLJcomplext, and/or wherein a lower expression of at least one polynucleotide defined by at least one ofthe numbers 1 to 50 of Table 2.46 having a negative fc value, and/or a higher expression of at least one polynucleotide defined by at least one ofthe numbers 1 to 50 of Table 2.46 having a positive fc value, is indicative for the presence AMLJVILL when AML_MLL distinguished from AML_t(15;17), and/or wherein a lower expression of at least one polynucleotide defined by at least one ofthe numbers 1 to 50 of Table 2.47 having a negative fc value, and/or a higher expression of at least one polynucleotide defined by at least one ofthe numbers 1 to 50 of Table 2.47 having a positive fc value, is indicative for the presence AMLJVILL when AMLJVILL distinguished from AML_t(8;21), and/or wherein a lower expression of at least one polynucleotide defined by at least one ofthe numbers 1 to 50 of Table 2.48 having a negative fc value, and/or a higher expression of at least one polynucleotide defined by at least one ofthe numbers 1 to 50 of Table 2.48 having a positive fc value, is indicative for the presence AMLJVILL when AMLJVILL distinguished from CLL, and/or wherein a lower expression of at least one polynucleotide defined by at least one ofthe numbers 1 to 50 of Table 2.49 having a negative fc value, and or a higher expression of at least one polynucleotide defined by at least one ofthe numbers 1 to 50 of Table 2.49 having a positive fc value, is indicative for the presence AMLJVILL when AMLJVILL distinguished from CML, and/or wherein a lower expression of at least one polynucleotide defined by at least one ofthe numbers 1 to 50 of Table 2.50 having a negative fc value, and/or a higher expression of at least one polynucleotide defined by at least one ofthe numbers 1 to 50 of Table 2.50 having a positive fc value, is indicative for the presence AMLJVILL when AMLJVILL distinguished from normal-BM, and/or wherein a lower expression of at least one polynucleotide defined by at least one ofthe numbers 1 to 50 of Table 2.51 having a negative fc value, and/or a higher expression of at least one polynucleotide defined by at least one ofthe numbers 1 to 50 of Table 2.51 having a positive fc value, is indicative for the presence AML_inv(16) when AML_inv(16) distinguished from AML_inv(3), and/or wherein a lower expression of at least one polynucleotide defined by at least one ofthe numbers 1 to 50 of Table 2.52 having a negative fc value, and/or a higher expression of at least one polynucleotide defined by at least one ofthe numbers 1 to 50 of Table 2.52 having a positive fc value, is indicative for the presence AML_inv(16) when AML_inv(16) distinguished from AMLJcomplext, and/or wherein a lower expression of at least one polynucleotide defined by at least one ofthe numbers 1 to 50 of Table 2.53 having a negative fc value, and/or a higher expression of at least one polynucleotide defined by at least one ofthe numbers 1 to 50 of Table 2.53 having a positive fc value, is indicative for the presence AML_inv( 16) when AML_inv( 16) distinguished from AML_t(l 5; 17), and/or wherein a lower expression of at least one polynucleotide defined by at least one ofthe numbers 1 to 50 of Table 2.54 having a negative fc value, and/or a higher expression of at least one polynucleotide defined by at least one ofthe numbers 1 to 50 of Table 2.54 having a positive fc value, is indicative for the presence AML_inv(16) when AML_inv(16) distinguished from AML_t(8;21), and/or wherein a lower expression of at least one polynucleotide defined by at least one ofthe numbers 1 to 50 of Table 2.55 having a negative fc value, and or a higher expression of at least one polynucleotide defined by at least one ofthe numbers 1 to 50 of Table 2.55 having a positive fc value, is indicative for the presence AML_inv(16) when AML_inv(16) distinguished from CLL, and/or wherein a lower expression of at least one polynucleotide defined by at least one ofthe numbers 1 to 50 of Table 2.56 having a negative fc value, and or a higher expression of at least one polynucleotide defined by at least one ofthe numbers 1 to 50 of Table 2.56 having a positive fc value, is indicative for the presence AML_inv( 16) when AML_inv( 16) distinguished from CML, and/or wherein a lower expression of at least one polynucleotide defined by at least one ofthe numbers 1 to 50 of Table 2.57 having a negative fc value, and/or a higher expression of at least one polynucleotide defined by at least one ofthe numbers 1 to 50 of Table 2.57 having a positive fc value, is indicative for the presence AML_inv(16) when AML_inv(16) distinguished from normal-BM, and/or wherein a lower expression of at least one polynucleotide defined by at least one ofthe numbers 1 to 50 of Table 2.58 having a negative fc value, and/or a higher expression of at least one polynucleotide defined by at least one ofthe numbers 1 to 50 of Table 2.58 having a positive fc value, is indicative for the presence AML_inv(3) when AML_inv(3) distinguished from AMLJcomplext, and/or wherein a lower expression of at least one polynucleotide defined by at least one ofthe numbers 1 to 50 of Table 2.59 having a negative fc value, and/or a higher expression of at least one polynucleotide defined by at least one ofthe numbers 1 to 50 of Table 2.59 having a positive fc value, is indicative for the presence AML_inv(3) when AML_inv(3) distinguished from AML_t(15;17), and/or wherein a lower expression of at least one polynucleotide defined by at least one ofthe numbers 1 to 50 of Table 2.60 having a negative fc value, and/or a higher expression of at least one polynucleotide defined by at least one ofthe numbers 1 to 50 of Table 2.60 having a positive fc value, is indicative for the presence AML_inv(3) when AML_inv(3) distinguished from AML_t(8.21), and/or wherein a lower expression of at least one polynucleotide defined by at least one ofthe numbers 1 to 50 of Table 2.61 having a negative fc value, and/or a higher expression of at least one polynucleotide defined by at least one ofthe numbers 1 to 50 of Table 2.61 having a positive fc value, is indicative for the presence AML_inv(3) when AML_inv(3) distinguished from CLL, and/or wherein a lower expression of at least one polynucleotide defined by at least one ofthe numbers 1 to 50 of Table 2.62 having a negative fc value, and/or a higher expression of at least one polynucleotide defined by at least one ofthe numbers 1 to 50 of Table 2.62 having a positive fc value, is indicative for the presence AML_inv(3) when AML_inv(3) distinguished from CML, and/or wherein a lower expression of at least one polynucleotide defined by at least one ofthe numbers 1 to 50 of Table 2.63 having a negative fc value, and/or a higher expression of at least one polynucleotide defined by at least one ofthe numbers 1 to 50 of Table 2.63 having a positive fc value, is indicative for the presence AML_inv(3) when AML_inv(3) distinguished from normal-BM, and/or wherein a lower expression of at least one polynucleotide defined by at least one ofthe numbers 1 to 50 of Table 2.64 having a negative fc value, and/or a higher expression of at least one polynucleotide defined by at least one ofthe numbers 1 to 50 of Table 2.64 having a positive fc value, is indicative for the presence AMLJcomplext when AMLJcomplext distinguished from AML_t(15;17), and/or wherein a lower expression of at least one polynucleotide defined by at least one ofthe numbers 1 to 50 of Table 2.65 having a negative fc value, and/or a higher expression of at least one polynucleotide defined by at least one ofthe numbers 1 to 50 of Table 2.65 having a positive fc value, is indicative for the presence AMLJcomplext when AMLJcomplext distinguished from AML_t(8;21), and/or wherein a lower expression of at least one polynucleotide defined by at least one ofthe numbers 1 to 50 of Table 2.66 having a negative fc value, and or a higher expression of at least one polynucleotide defined by at least one ofthe numbers 1 to 50 of Table 2.66 having a positive fc value, is indicative for the presence AMLJcomplext when AMLJcomplext distinguished from CLL, and/or wherein a lower expression of at least one polynucleotide defined by at least one ofthe numbers 1 to 50 of Table 2.67 having a negative fc value, and/or a higher expression of at least one polynucleotide defined by at least one ofthe numbers 1 to 50 of Table 2.67 having a positive fc value, is indicative for the presence AJVILJcomplext when AMLJcomplext distinguished from CML, and/or wherein a lower expression of at least one polynucleotide defined by at least one ofthe numbers 1 to 50 of Table 2.68 having a negative fc value, and/or a higher expression of at least one polynucleotide defined by at least one ofthe numbers 1 to 50 of Table 2.68 having a positive fc value, is indicative for the presence AMLJcomplext when AMLJcomplext distinguished from normal-BM, and/or wherein a lower expression of at least one polynucleotide defined by at least one ofthe numbers 1 to 50 of Table 2.69 having a negative fc value, and/or a higher expression of at least one polynucleotide defined by at least one ofthe numbers 1 to 50 of Table 2.69 having a positive fc value, is indicative for the presence AML_t( 15 ; 17) when AML_t( 15 ; 17) is distinguished from AML_t(8;21), and/or wherein a lower expression of at least one polynucleotide defined by at least one ofthe numbers 1 to 50 of Table 2.70 having a negative fc value, and/or a higher expression of at least one polynucleotide defined by at least one ofthe numbers 1 to 50 of Table 2.70 having a positive fc value, is indicative for the presence AML_t(15;17) when AML_t(15;17) is distinguished from CLL, and/or wherein a lower expression of at least one polynucleotide defined by at least one ofthe numbers 1 to 50 of Table 2.71 having a negative fc value, and/or a higher expression of at least one polynucleotide defined by at least one ofthe numbers 1 to 50 of Table 2.71 having a positive fc value, is indicative for the presence AML_t(15;17) when AML_t(15;17) is distinguished from CML, and/or wherein a lower expression of at least one polynucleotide defined by at least one ofthe numbers 1 to 50 of Table 2.72 having a negative fc value, and/or a higher expression of at least one polynucleotide defined by at least one ofthe numbers 1 to 50 of Table 2.72 having a positive fc value, is indicative for the presence AML_t(15;17) when AML_t(15;17) is distinguished from normal-BM, and/or wherein a lower expression of at least one polynucleotide defined by at least one ofthe numbers 1 to 50 of Table 2.73 having a negative fc value, and/or a higher expression of at least one polynucleotide defined by at least one ofthe numbers 1 to 50 of Table 2.73 having a positive fc value, is indicative for the presence AML_t(8;21) when AML_t(8;21) is distinguished from CLL, and/or wherein a lower expression of at least one polynucleotide defined by at least one ofthe numbers 1 to 50 of Table 2.74 having a negative fc value, and/or a higher expression of at least one polynucleotide defined by at least one ofthe numbers 1 to 50 of Table 2.74 having a positive fc value, is indicative for the presence AML_t(8;21) when AML_t(8;21) is distinguished from CML, and/or wherein a lower expression of at least one polynucleotide defined by at least one ofthe numbers 1 to 50 of Table 2.75 having a negative fc value, and/or a higher expression of at least one polynucleotide defined by at least one ofthe numbers 1 to 50 of Table 2.75 having a positive fc value, is indicative for the presence AML_t(8;21) when AML_t(8;21) is distinguished from normal-BM, and/or wherein a lower expression of at least one polynucleotide defined by at least one ofthe numbers 1 to 50 of Table 2.76 having a negative fc value, and/or a higher expression of at least one polynucleotide defined by at least one ofthe numbers 1 to 50 of Table 2.76 having a positive fc value, is indicative for the presence CLL when CLL is distinguished from CML, and/or wherein a lower expression of at least one polynucleotide defined by at least one ofthe numbers 1 to 50 of Table 2.77 having a negative fc value, and/or a higher expression of at least one polynucleotide defined by at least one ofthe numbers 1 to 50 of Table 2.77 having a positive fc value, is indicative for the presence CLL when CLL is distinguished from normal-BM, and/or wherein a lower expression of at least one polynucleotide defined by at least one ofthe numbers 1 to 50 of Table 2.78 having a negative fc value, and/or a higher expression of at least one polynucleotide defined by at least one ofthe numbers 1 to 50 of Table 2.78 having a positive fc value, is indicative for the presence CML when CML is distinguished from normal-BM.
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 ofthe 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 ofthe markers of at least one ofthe Tables 1.1- 2.78 is determined.
5. The method according to at least one ofthe 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 ofthe 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 ofthe claims 1-6, wherein the sample is from an individual having leukemia.
8. The method according to at least one ofthe 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 mRNA or a cDNA.
10. The method according to claim 8 or 9, wherein the determining ofthe 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 ofthe 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 ofthe 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 ofthe claims 1-13, wherein the method is carried out on an array.
15. The method according to at least one ofthe claims 1-14, wherein the method is carried out in a robotics system.
16. The method according to at least one ofthe claims 1-15, wherein the method is carried out using microfluidics.
17. Use of at least one marker as defined in at least one ofthe claims 1-3 for the manufacturing of a diagnostic for distinguishing leukemia subtypes t(15;17), t(8;21), inv(16), inv(3), complex aberrant karyotype (CA), AML- MLL, normal karyotype (NK), AML-other (trisomy 8, trisomy 11, trisomy 13, monosomy 7, del(5q), del(9q), t(6;9); del(20q) and del(12p) and trisomy 4), ALL-MLL, ALL-Ph+, ALL-t(8; 14), T-ALL, other B-lineage (OBL), CML, normal-BM, and/or CLL.
18. The use according to claim 17 for distinguishing leukemia subtypes t(15;17), t(8;21), inv(16), inv(3), complex aberrant karyotype (CA), AML- MLL, normal karyotype (NK), AML-other (trisomy 8, trisomy 11, trisomy 13, monosomy 7, del(5q), del(9q), t(6;9); del(20q) and del(12p) and trisomy 4), ALL-MLL, ALL-Ph+, ALL-t(8;14), T-ALL, other B-lineage (OBL), CML, normal-BM, and/or CLL in an individual having leukemia.
19. A diagnostic kit containing at least one marker as defined in at least one of the claims 1-3 for distinguishing leukemia subtypes t(15;17), t(8;21), inv(16), inv(3), complex aberrant karyotype (CA), AML-MLL, normal karyotype (NK), AML-other (trisomy 8, trisomy 11, trisomy 13, monosomy 7, del(5q), del(9q), t(6;9); del(20q) and del(12p) and trisomy 4), ALL-MLL, ALL-Ph+, ALL-t(8; 14), T-ALL, other B-lineage (OBL), CML, normal-BM, and/or CLL, in combination with suitable auxiliaries.
20. The diagnostic kit according to claim 19, wherein the kit contains a reference for the leukemia subtypes t(15;17), t(8;21), inv(16), inv(3), complex aberrant karyotype (CA), AML-MLL, normal karyotype (NK), AML-other (trisomy 8, trisomy 11 , trisomy 13, monosomy 7, del(5q), del(9q), t(6;9); del(20q) and del(12p) and trisomy 4), ALL-MLL, ALL-Ph+, ALL-t(8; 14), T-ALL, other B-lineage (OBL), CML, normal-BM, and/or CLL.
21. The diagnostic kit according to claim 20, wherein the reference is a sample or a data bank.
22. An apparatus for distinguishing leukemia subtypes t(15;17), t(8;21), inv(16), inv(3), complex aberrant karyotype (CA), AML-MLL, normal karyotype (NK), AML-other (trisomy 8, trisomy 11, trisomy 13, monosomy 7, del(5q), del(9q), t(6;9); del(20q) and del(12p) and trisomy 4), ALL-MLL, ALL-Ph+, ALL-t(8;14), T-ALL, other B-lineage (OBL), CML, normal-BM, and or CLL 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, and/or 2, 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 Voting, 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 ofthe claims 22-24, wherein the apparatus contains a control panel and/or a monitor.
26. A reference data bank for distinguishing leukemia subtypes t(15;17), t(8;21), inv(16), inv(3), complex aberrant karyotype (CA), AML-MLL, normal karyotype (NK), AML-other (trisomy 8, trisomy 11, trisomy 13, monosomy 7, del(5q), del(9q), t(6;9); del(20q) and del(12p) and trisomy A), ALL-MLL, ALL-Ph+, ALL-t(8;14), T-ALL, other B-lineage (OBL), CML, normal-BM, and/or CLL 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 2, 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.
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EP2993473A1 (en) * 2007-01-30 2016-03-09 Pharmacyclics, Inc. Methods for determining cancer resistance to histone deacetylase inhibitors
US9492423B2 (en) 2011-09-13 2016-11-15 Pharmacyclics Llc Formulations of histone deacetylase inhibitor in combination with bendamustine and uses thereof

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