US 20120070450A1 (19) United States (12) Patent Application Publication (10) Pub. No.: US 2012/0070450 A1 Ishikawa et al. (43) Pub. Date: Mar. 22, 2012

(54) LEUKEMA STEM CELLMARKERS Publication Classification (51) Int. Cl. A 6LX 39/395 (2006.01) (75) Inventors: Fumihiko Ishikawa, Kanagawa CI2O I/68 (2006.01) (JP): Osamu Ohara, Kanagawa GOIN 2L/64 (2006.01) (JP); Yoriko Saito, Kanagawa (JP); A6IP35/02 (2006.01) Hiroshi Kitamura, Kanagawa (JP); C40B 30/04 (2006.01) Atsushi Hijikata, Kanagawa (JP); A63L/7088 (2006.01) Hidetoshi Ozawa, Kanagawa (JP); C07K 6/8 (2006.01) Leonard D. Shultz, Bar Harbor, C7H 2L/00 (2006.01) A6II 35/12 (2006.01) ME (US) CI2N 5/078 (2010.01) (52) U.S. Cl...... 424/173.1; 424/178.1; 424/93.7: (73) Assignee: RIKEN, Wako-shi (JP) 435/6.14; 435/723; 435/375; 506/9: 514/44 A: 530/389.6; 530/391.7:536/24.5 (57) ABSTRACT (21) Appl. No.: 13/258,993 The invention provides a test method for predicting the initial onset or a recurrence of acute myeloid (AML) com PCT Fled: prising (1) measuring the expression level of human leukemic (22) Mar. 24, 2010 stem cell (LSC) marker in a biological sample collected from a Subject for a transcription product or translation prod uct of the as an analyte and (2) comparing the expression (86) PCT NO.: PCT/UP2010/0551.31 level with a reference value; an LSC-targeting therapeutic agent for AML capable of Suppressing the expression of a S371 (c)(1), gene selected from among LSC marker genes or a Substance (2), (4) Date: Dec. 7, 2011 capable of suppressing the activity of a translation product of the gene; a method for producing a sample containing hematopoietic cells for autologous transplantation or alloge (30) Foreign Application Priority Data neic transplantation for AML patients comprising obtaining an LSC-purged sample with at least 1 kind of LSC marker as Mar. 24, 2009 (JP) ...... 2009-072400 an index; and a method of preventing or treating AML. Patent Application Publication US 2012/0070450 A1

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LEUKEMLA STEM CELLMARKERS hematopoiesis: are a stem cell disorder or a reacquisition of stem cell characteristics? Proc Natl Acad TECHNICAL FIELD Sci USA 100 Suppl 1, 11842-11849 (2003). 0001. The present invention relates to leukemic stem cell 0006 non-patent document 2: Hope, K.J., Jin, L. & Dick, markers and the field of treatment of acute myeloid leukemia. J. E. Acute myeloid leukemia originates from a hierarchy of leukemic stem cell classes that differ in self-renewal BACKGROUND ART capacity. Nat Immunol 5, 738-743 (2004). 0002 Acute myeloid leukemia (AML) is the most com 0007 non-patent document 3: Jordan, C. T. & Guzman, mon/highly frequent (onset rate) adult leukemia, character M. L. Mechanisms controlling pathogenesis and Survival ized by the clonal expansion of immature myeloblasts initi of leukemic stem cells. Oncogene 23, 7178-7187 (2004). ating from rare leukemic stem cells (LSCs) (non-patent 0008 non-patent document 4: Lapidot, T. et al. A cell documents 1-3). The functional and molecular characteristics initiating human acute myeloid leukaemia after transplan of human LSCs are largely undetermined. Although conven tation into SCID mice. Nature 367, 645-648 (1994). tional chemotherapeutic agents can temporarily remit AML. 0009 non-patent document 5: Ishikawa, F. et al. Chemo recurrence later is the difficult problem that prevents us from therapy-resistant human AML stem cells home to and helping patients. For the development of an effective thera engraft within the bone marrow endosteal region. Nature peutic agent or treatment method, elucidation of the recur Biotechnol 25:1315-1321 (2007). rence mechanism by clarifying the leukemia features 0010 non-patent document 6: Clarke, M. F. et al. Cancer unknown to date is strongly desired. stem cells perspectives on current status and future direc 0003. A recent study demonstrated that a certain ratio of tions: AACR Workshop on cancer stem cells. Cancer Res leukemias and cancers consists of a heterogenous cell frac 66,9339-9344 (2006). tion and is not configured with a homogenous cell population capable of clonal proliferation. Lapidot and Dick identified SUMMARY OF THE INVENTION Such heterogeneity in acute myeloid leukemia (AML) and Problems to Be Solved by the Invention reported that CD34+CD38-cells are transplanted selectively in CB17-scid and NOD/SCID mice (Non-patent Document 0011 A problem to be solved is to find a molecular target 4). that is specific for human leukemic stem cells (LSCs) and 0004. The present inventors have succeeded in the devel provide a therapeutic means that will lead to radical treatment opment of an animal model capable of reproducing features of acute myeloid leukemia (AML) and the like. of human, rather than mouse, AML, particularly AML of individual patients, rather than a cell line, and permitting Means of Solving the Problems long-term assessment (Non-patent Document 5, Patent 0012. The present inventors found sets of genes differen Application PCT/JP2008/068892). The present inventors fur tially expressed between LSCs and non-stem cells, and pro ther identified using a neonatal NOD/SCID/IL2rg KO mouse posed the possibility that these genes serve as therapeutic model, which is one of the most sensitive human stem cell targets for AML (Ishikawa F. et al., Nature Biotechnol assays, that CD34+CD38-AML cells meet all criteria for 25:1315-1321, 2007 and PCT/JP2008/068892), but were cancer stem cells recommended by the American Association unable to rule out the possibility that the genes are at the same for Cancer Research (Non-patent Document 6). Specifically, time differentially expressed in normal hematopoietic stem CD34+CD38-AML cells self-renew, produce non-stem leu cells (HSCs) as well. Hence, a therapeutic agent and thera kemia cells, and have the exclusive capability of causing peutic method for AML with low prevalence of adverse reac AML in living organisms. By repeating primary human AML tions cannot be realized unless not only a comparison is made in NOD/SCID/IL2rg KO mice, the present inventors searched between LSCs and non-stem cells, but also a set of genes that for the mechanism behind the chemotherapy resistance and are differentially expressed between LSCs and HSCs are recurrences, which pose the most important problem in the identified as targets. The present inventors succeeded in reality of this disease, and identified the following two essen developing a mouse model enabling reproduction of human tial features of human AML stem cells. First, AML stem cells AML (mice generated by transplanting a fraction containing are present predominantly in the endosteal region of the bone leukemic stem cells derived from a human AML patient to marrow; when human AML transplantation recipient mice NOD/SCID/IL2rg" mice), transplanting a small number of were treated with chemotherapeutic agents, the great major bone marrow cells derived from an AML patient, and recon ity of chemotherapy-resistant AML cells were found in osteo structing the pathology of AML in the animal model. The blast niches. Second, AML stem cells (not CD34+CD38+and present inventors then prepared LSCs derived from an AML CD34-AML cells) are stationary and hence exhibit resistance patient and those from an AML transplantation recipient to cell cycle-dependent chemotherapeutic agents. These his mouse, as well as bone marrow samples and cord blood tological experiments and cell cycle analyses agree with the samples (HSCs are contained) derived from healthy donors, clinical evidence that a large number of AML patients achieve conducted a comprehensive analysis, and have developed the remission via chemotherapy induction but eventually experi present invention. ence recurrences. To develop a novel therapeutic strategy 0013. Accordingly, the present invention provides the fol designed to exterminate LSCs seems to be an exact step lowing. toward overcoming recurrences of AML. 1 A test method for predicting the initial onset or a recur Prior Art References rence of acute myeloid leukemia, comprising (1) a step of measuring the expression level of leukemic stem Non-Patent Documents cell marker genes in a biological sample collected from a 0005 non-patent document 1: Passegue, E., Jamieson, C. Subject for a transcription product or translation product of H., Ailles, L. E. & Weissman, I. L. Normal and leukemic the genes as an analyte, and

US 2012/0070450 A1 Mar. 22, 2012

naling-related genes consisting of AK5, ARHGAP18. FYB, PDE9A, PDK1, PRKAR1A, PRKCD, PXK, RAB20, HCK, LPXN, PDE9A, PDK1, PRKCD, RAB20, RAB8A and RAB8A, RABIF, RASGRP3, RGS18 and S100A11; RABIF; transcription factors consisting of WT1 and HLX; transcription factor genes consisting of WT1, MYC and and other genes consisting of CYBB, CTSC and NCF4. HLX; and 5 The therapeutic agent according to 3, wherein the leu other genes consisting of ACTR2. ALOX5, ANXA2P2, kemic stem cell marker gene is selected from the group con ATL3, ATP6V1B2, ATP6V1C1, ATP6V1D, C12orf5, sisting of C17orf60, C18orf19, C1GALT1C1, C1orf135, C1orf163, cell membrane- or extracellularly-localized genes consisting C1orf186, C60rf150, CALML4, CCT5, CLC, COMMD8, of ALOX5AP CACNB4, CCL5, CD33, CD3D, CD93, COTL1, COX17, CRIP1, CSTA, CTSA, CTSC, CTSG, CD97, CLEC12A, DOK2, FCGR2A, GPR84, HCST, CYBB, CYP2E1, DENND3, DHRS3, DLAT, DLEU2, HOMER3, ITGB2, LGALS1, LRG1, PTH2R, RNASE2, DPH3, EFHD2, ENC1, EXOSC3, FAM107B, FAM129A, TNF, TNFSF13B, TYROBP and VNN1; a cell cycle-related FAM38B, FBXO22, FLJ14213, FNDC3B, GNPDA1, gene consisting of NEK6; an apoptosis-related gene consist GRPEL1, GTSF1, HIG2, HN1, HVCN1, IDH1, IDH3A, ing of BIK; signaling-related genes consisting of AK5, FYB, IKIP, KIF2C, KYNU, LCMT2, ME1, MIRN21, MKKS, HCK, LPXN, PDE9A, PDK1, PRKCD and RAB20; a tran MNDA, MTHFD2, MYO1B, MYO1F, NAGA, NCF2, Scription factor gene consisting of WT1; and other genes NCF4, NDUFAF1, NP, NRIP3, OBFC2A, PARP8, PDLIM1, consisting of CTSC and NCF4. PDSS1, PGM2, PIGK, PIWIL4, PPCDC, PPIF, PRAME, 6. The therapeutic agent according to 3, wherein the leu PUS7, RPP40, RRM2, S100A16, S100A8, S100P, S100Z, kemic stem cell marker gene is a marker expressed in stem SAMHD1, SH2D1A, SPCS2, SPPL2A, TESC, THEX1, cells that are present in bone marrow niches, are in the sta TMEM30A, TMEM33, TRIP13, TUBB6, UBASH3B, tionary phase of cell cycle, and are resistant to anticancer UGCG, VSTM1, WDR4, WIT1, WSB2 and ZNF253; and agents, selected from the group consisting of AK5, BIK, 0018 c) a step of sorting cells to which the substance has DOK2, FCGR2A, IL2RA, LRG1, SUCNR1 and WT1. bound, and obtaining the sample from which leukemic stem 7. The therapeutic agent according to any one of 3 to 6. cells have been purged. wherein the Substance capable of Suppressing the expression 11 The production method according to 10, wherein the of the gene is an antisense nucleic acid oran RNAi-inducible leukemic stem cell marker is at least one kind of cell Surface nucleic acid. marker gene selected from among ADFP. ALOX5AP. 8. The therapeutic agent according to any one of 3 to 6. CACNB4, CD33, CD3D, CD93, CD97, CLEC12A, DOK2, wherein the substance capable of suppressing the activity of a FCER1G, FCGR2A, GPR34, GPR84, HCST, HOMER3, translation product is an aptamer or an antibody. IL2RA, IL2RG, IL3RA, ITGB2, LY86, P2RY5, PTH2R, 9. The therapeutic agent according to 8, wherein the anti SUCNR1, TNFRSF4, TYROBP and VNN1. body is an immunoconjugate of an antibody and an anticancer 12 A method for preventing or treating acute myeloid leu Substance. kemia that targets leukemic stem cells, comprising adminis 10A production method of a sample containing hematopoi tering, to a subject, an effective amount of a Substance capable etic cells for autologous transplantation or allogeneic trans of suppressing the expression of a gene selected from among plantation for a patient with acute myeloid leukemia, com leukemic stem cell marker genes consisting of the following prising: set of genes: 0016 a) a step of collecting a sample containing hemato cell membrane- or extracellularly-localized genes consisting poietic cells from the patient or a donor, of ADFP. ALOX5AP, AZU1, C3AR1, CACNB4, CALCRL, 0017 b) a step of bringing the collected sample into con CCL4, COL5, CD33, CD36, CD3D, CD86, CD9, CD93, tact with a Substance that recognizes a translation product of CD96, CD97, CFD, CHI3L1, CLEC12A, CLECL1, COCH, at least one kind of leukemic stem cell marker gene selected CST7, CXCL1, DOK2, EMR2, FCER1G, FCGR2A, from among the following set of genes: FUCA2, GPR109B, GPR160, GPR34, GPR84, HAVCR2, cell membrane- or extracellularly-localized genes consisting HBEGF, HCST, HGF, HLA-DOB, HOMER3, IFI30, of ADFP. ALOX5AP, AZU1, C3AR1, CACNB4, CALCRL, IL13RA1, IL2RA, IL2RG, IL3RA, INHBA, ITGB2, CCL4, CCL5, CD33, CD36, CD3D, CD86, CD9, CD93, LGALS1, LRG1, LY86, MAMDC2, MGAT4A, P2RY14, CD96, CD97, CFD, CHI3L1, CLEC12A, CLECL1, COCH, P2RY5, PLAUR, PPBP, PRG2, PRSS21, PTH2R, PTX3, CST7, CXCL1, DOK2, EMR2, FCER1G, FCGR2A, REEP5, RNASE2, RXFP1, SLC31A2, SLC43A3, SLC6A6, FUCA2, GPR109B, GPR160, GPR34, GPR84, HAVCR2, SLC7A6, STX7, SUCNR1, TACSTD2, TIMP1, TM4SF1, HBEGF, HCST, HGF, HLA-DOB, HOMER3, IFI30, TM9SF1, TNF, TNFRSF4, TNFSF13B, TYROBP, UTS2 and IL13RA1, IL2RA, TL2RG, IL3RA, INHEA, ITGB2, VNN1; LGALS1, LRG1, LY86, MAMDC2, MGAT4A, P2RY14, cell cycle-related genes consisting of AURKA, C13orf34, P2RY5, PLAUR, PPBP, PRG2, PRSS21, PTH2R, PTX3, CCNA1, DSCC1, FAM33A, HPGD, NEK6, PYHIN1, REEP5, RNASE2, RXFP1, SLC31A2, SLC43A3, SLC6A6, RASSF4, TXNL4B and ZWINT: SLC7A6, STX7, SUCNR1, TACSTD2, TIMP1, TM4SF1, apoptosis-related genes consisting of MPO, IER3, BIK, TM9SF1, TNF, TNFRSF4, TNFSF13B, TYROBP, UTS2 and TXNDC1, GADD45B and NAIP; VNN1; signaling-related genes consisting of AK5, ARHGAP18. cell cycle-related genes consisting of AURKA, C13orf34, ARRB1, DUSP6, FYB, HCK, LPXN, MS4A3, PAK1IP1, CCNA1, DSCC1, FAM33A, HPGD, NEK6, PYHIN1, PDE9A, PDK1, PRKAR1A, PRKCD, PXK, RAB20, RASSF4, TXNL4B and ZWINT: apoptosis-related genes RAB8A, RABIF, RASGRP3, RGS18 and S100A11; consisting of MPO, IER3, BIK, TXNDC1, GADD45B and transcription factor genes consisting of WT1, MYC and NAIP; HLX; and signaling-related genes consisting of AK5, ARHGAP18. other genes consisting of ACTR2. ALOX5, ANXA2P2, ARRB1, DUSP6, FYB, HCK, LPXN, MS4A3, PAK1IP1, ATL3, ATP6V1B2, ATP6V1C1, ATP6V1D, C12orf5, US 2012/0070450 A1 Mar. 22, 2012

C17orf60, C18orf19, C1GALT1C1, C1orf135, C1orf163, 0023 FIG. 2 shows genes expressed in larger amounts in C1orf186, C60rf150, CALML4, CCT5, CLC, COMMD8, AML CD34+CD38- LSCs than in normal CD34+CD38 COTL1, COX17, CRIP1, CSTA, CTSA, CTSC, CTSG, HSCs. The heat map includes qPCR data on 35 prominent CYBB, CYP2E1, DENND3, DHRS3, DLAT, DLEU2, LSC markers: 1) their functions and localization are suitable DPH3, EFHD2, ENC1, EXOSC3, FAM107B, FAM129A, for the development of anti-AML drugs, 2) their mRNA con FAM38B, FBXO22, FLJ14213, FNDC3B, GNPDA1, tents are significantly (P<0.05) higher in LSCs than in HSCs, GRPEL1, GTSF1, HIG2, HN1, HVCN1, IDH1, IDH3A, 3) the median of their mRNA contents are 5 times or more IKIP, KIF2C, KYNU, LCMT2, ME1, MIRN21, MKKS, higher in LSCs than in HSCs, and 4) their mRNA contents are MNDA, MTHFD2, MYO1B, MYO1F, NAGA, NCF2, higher in all LSC samples tested than in various HSC NCF4, NDUFAF1, NP, NRIP3, OBFC2A, PARP8, PDLIM1, samples. In this panel, red, yellow and green indicate high, PDSS1, PGM2, PIGK, PIWIL4, PPCDC, PPIF, PRAME, moderate, and low expression, respectively, as shown by the PUS7, RPP40, RRM2, S100A16, S100A8, S100P, S100Z, reference color code in the lower left in this figure. Value 1 SAMHD1, SH2D1A, SPCS2, SPPL2A, TESC, THEX1, indicates the mean for mRNAS in CD34+CD38- HSCs. TMEM30A, TMEM33, TRIP13, TUBB6, UBASH3B, 0024 FIG. 3 shows flow cytometry. The expression of UGCG, VSTM1, WDR4, WIT4, WSB2 and ZNF253; LSC-specific molecule candidates (CD32, ITGB2, CD93 and or a substance capable of Suppressing the activity of a trans CD33) was analyzed by flow cytometry. Each histogram lation product of the gene. shows relative expression in LSCs obtained from five AML patients versus that in normal HSCs. Effect of the Invention 0025 FIG. 4 shows the results of functional assay and histological experiments of CD32. The expression of CD32 0019. The present invention has been developed as a result and the expression of CD133 were again analyzed by FACS. of Succeeding in analyzing the comprehensive expression According to the expression pattern of CD32, AML patients profiling of leukemic stem cells (LSCs) derived from human were classified under the categories AML-a and AML-b. Nor primary AML, and identifying LSC-specific targets for sepa mal HSCs were identified exclusively in the CD32-fraction. rating LSCs from HSCs. Therefore, the leukemic stem cell Likewise, leukemia induction activity was observed in the markers found in the present invention make it possible not CD32-fraction of the AML-a group. In contrast, in AML-b. only to distinguish between non-stem cells and LSCs, but also CD32+ cells exhibited the capability of initiating AML in to distinguish between normal hematopoietic stem cells vivo. In AML-b, CD32+ cells were detected in both the mem (HSCs) and LSCs, which have been thought to be difficult to brane region and central region of the bone marrow. distinguish from each other. By using a leukemic stem cell 0026 FIG. 5 shows heat map charts of gene candidates marker found in the present invention as a molecular target, a whose transcription products are more highly expressed in therapeutic agent that acts specifically on LSCs that are the AML CD34+CD38- LSCs than in normal CD34+CD38 source of onset or recurrence of AML can be provided. HSCs. 217 genes were classified on the basis of gene ontol 0020. Also, it is possible to specifically remove LSCs from ogy under six categories: 1) cell membrane and extracellular, bone marrow cells of a patient or a donor using a cell sorter 2) cell cycle, 3) apoptosis, 4) signaling, 5) transcription fac such as FACS, with a leukemic stem cell marker found in the tors, and 6) others. Gene expression levels on two microarray present invention as an index. This will lead to the effective platforms (U133 plus 2.0 and Gene 1.0ST) are separately removal of the true source of onset or recurrences of AML. shown. In each panel, red, yellow and green indicate high, Therefore, recurrences of AML can be prevented signifi moderate and low expression, respectively. cantly. 0027 FIG. 6 is a flow cytometric representation showing 0021. Furthermore, the presence or absence of LSCs in a that the expression of CD32, one of the above-described collected biological sample or in a body can be determined candidate genes, does not undergo down regulation in AML with a leukemic stem cell marker found in the present inven patients after chemotherapy. tion as an index, whereby recurrences or the initial onset of 0028 FIG. 7 shows immunofluorescent staining of the acute myeloid leukemia can also be predicted. expression of various marker genes in leukemic stem cells that are present in bone marrow niches and are in the station BRIEF DESCRIPTION OF THE DRAWINGS ary phase of cell cycle. The results for each gene are shown 0022 FIG. 1 shows the results of transplantation of normal with a set of four photographs obtained using the DAPI anti CD34+CD38-HSCs and AMLCD34+CD38-LSCs. (Upper body (nuclear staining) for blue staining in the upper left, an panel) Transplantation of normal CD34+CD38- cells antibody against the marker for red staining in the lower left, resulted in efficient reconstitution of human CD45+ hemato and an antibody against the cell cycle marker CD34 (in the poietic cells. Because differentiation into normal human case of FCGR2A, AK5, DOK2, LRG1, BIK) or the Ki67 immunocytes such as CD11c-- ordinary dendritic cells, antibody (in the case of IL2RA, WT1, SUCNR1) for green CD123-high plasmacytoid dendritic cells, T cells and B cells staining in the upper right. Shown in the lower right are is observed in human CD45+ cells, it is seen that the CD34+ merged results. CD38- are hematopoietic stem cells. (Lower panel) When AML CD34+CD38-cells were transplanted, AML devel MODES FOR EMBODYING THE INVENTION oped in recipient mice. Recipient BM was completely occu Definitions pied by human CD45+ cells, rather than by mouse cells. Because the transplanted human cells did not contain any of 0029. In the present invention, the initial onset of leukemia normal immunocyte Subsets such as dendritic cells, T cells or refers to a state in which leukemia has developed for the first B cells, the CD34+CD38-cells were shown to contain no time, or is likely to develop, and a recurrence of leukemia normal hematopoietic stem cells and were identified as leu refers to a state in which leukemia has developed again, or is kemic stem cells. likely to develop, after treatment or remission of initial-onset US 2012/0070450 A1 Mar. 22, 2012

leukemia. The tissue where leukemia recurs or is likely to CCL4, CCL5, CD33, CD36, CD3D, CD86, CD9, CD93, recur is not limited to initial-onset tissue, and may be another CD96, CD97, CFD, CHI3L1, CLEC12A, CLECL1, COCH, tissue. Therefore, the concept of recurrence is understood to CST7, CXCL1, DOK2, EMR2, FCER1G, FCGR2A, include infiltration and metastasis. FUCA2, GPR109B, GPR160, GPR34, GPR84, HAVCR2, 0030. In the present invention, treatment of leukemia HBEGF, HCST, HGF, HLA-DOB, HOMER3, IFI30, encompasses all treatments, including administration of anti IL13RA1, IL2RA, IL2RG, IL3RA, INHBA, ITGB2, cancer agents, radiotherapy, and bone marrow transplanta LGALS1, LRG1, LY86, MAMDC2, MGAT4A, P2RY14, tion. P2RY5, PLAUR, PPBP, PRG2, PRSS21, PTH2R, PTX3, 0031. In the present invention, leukemic stem cells (LSC) REEP5, RNASE2, RXFP1, SLC31A2, SLC43A3, SLC6A6, may be a CD34+ cell fraction derived from the bone marrow, SLC7A6, STX7, SUCNR1, TACSTD2, TIMP1, TM4SF1, with preference given to CD34+CD38-cell fraction. The TM9SF1, TNF, TNFRSF4, TNFSF13B, TYROBP, UTS2 and crude substance containing LSC can be recovered from the VNN1; bone marrow of a test Subject or patient by a conventional cell cycle-related genes consisting of AURKA, C13orf34, method, cell fractions containing the LSC can be obtained by CCNA1, DSCC1, FAM33A, HPGD, NEK6, PYHIN1, flow cytometry and the like using CD34 and CD38 cell sur RASSF4, TXNL4B and ZWINT: apoptosis-related genes face marker molecules. Note that separation of LSC from consisting of MPO, IER3, BIK, TXNDC1, GADD45B and HSC is difficult. Furthermore, it is also possible to further sort NAIP; LSCs with another cell surface marker molecule selected signaling-related genes consisting of AK5, ARHGAP18. from among leukemic stem cell markers found by the present ARRB1, DUSP6, FYB, HCK, LPXN, MS4A3, PAK1IP1, invention, as an index. PDE9A, PDK1, PRKAR1A, PRKCD, PXK, RAB20, RAB8A, RABIF, RASGRP3, RGS18 and S100A11; (Test Method) transcription factor genes consisting of WT1, MYC and HLX; and 0032. The present invention provides a test method for other genes consisting of ACTR2. ALOX5, ANXA2P2, predicting the initial onset or a recurrence of acute myeloid ATL3, ATP6V1B2, ATP6V1C1, ATP6V1D, C12orf5, leukemia. The test method of the present invention comprises, C17orf60, C18orf19, C1GALT1C1, C1orf135, C1orf163, (1) a step of measuring the expression level of leukemic stem C1orf186, C60rf150, CALML4, CCT5, CLC, COMMD8, cell marker genes in a biological sample collected from a COTL1, COX17, CRIP1, CSTA, CTSA, CTSC, CTSG, Subject for a transcription product or translation product of CYBB, CYP2E1, DENND3, DHRS3, DLAT, DLEU2, the gene as an analyte, and DPH3, EFHD2, ENC1, EXOSC3, FAM107B, FAM129A, (2) a step of comparing the expression levels obtained in the FAM38B, FBXO22, FLJ14213, FNDC3B, GNPDA1, measuring step with the expression level in healthy persons. GRPEL1, GTSF1, HIG2, HN1, HVCN1, IDH1, IDH3A, (1) Step of measuring the expression level of leukemic stem IKIP, KIF2C, KYNU, LCMT2, ME1, MIRN21, MKKS, cell marker genes in a biological sample collected from a MNDA, MTHFD2, MYO1B, MYO1F, NAGA, NCF2, Subject for a transcription product or translation product of NCF4, NDUFAF1, NP, NRIP3, OBFC2A, PARP8, PDLIM1, the gene as an analyte PDSS1, PGM2, PIGK, PIWIL4, PPCDC, PPIF, PRAME, 0033) Leukemic stem cell marker genes targeted in the PUS7, RPP40, RRM2, S100A16, S100A8, S100P, S100Z, present invention are leukemic stem cell-specific markers SAMHD1, SH2D1A, SPCS2, SPPL2A, TESC, THEX1, sorted from a set of genes expressed differentially in the TMEM30A, TMEM33, TRIP13, TUBB6, UBASH3B, CD34+CD38- cell fraction than in the CD34+CD38+ cell UGCG, VSTM1, WDR4, WIT1, WSB2 and ZNF253. fraction by the present inventors on the basis of their unique 0035. The individual genes that constitute the aforemen viewpoint, and comprise 2 to 218 genes selected from among tioned leukemic stem cell marker genes are publicly known, the following leukemic stem cell marker genes (hereinafter and the base sequences and amino acid sequences thereofare sometimes simply abbreviated as “marker genes' or “mark also known. ers') (1). The marker genes (1) preferably consist of 3 or 0036. For the marker genes except IL2RA, symbol names, more, 5 or more, 10 or more, 15 or more, 20 or more, or 25 or gene IDs, location , characteristics and the like more, genes. are shown in Table 1. IL2RA, also called CD25 has the gene 0034 Marker Genes (1): ID 3559, is located on 10, and encodes interleu cell membrane- or extracellularly-localized genes consisting kin 2 receptor alpha. The IL2RA is a transmembra of ADFP. ALOX5AP, AZU1, C3AR1, CACNB4, CALCRL, nous receptor localized on the cell membrane.

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0037. When the test method of the present invention is and proteases obtained from their manufacturers, such as intended to more clearly distinguish between LSCs and Qiagen, as directed by the manufacturers. HSCs, it is preferable that the following marker genes (2), for 0043. The method of measuring the expression level of a example, out of the above-described marker genes (1), be marker gene for a transcription product as an analyte is not used as an index. In this mode of embodiment, the marker particularly limited; available methods include Northern genes (2) consist of 2 to 58 genes, more preferably consist of blotting and in situ hybridization (Parker & Barnes, Methods 3 or more, or more, 10 or more, 15 or more, 20 or more, or 25 in Molecular Biology 106: 247-283 (1999)); RNase protec or more, genes. When the test method of the present invention tion assay (Hod, Biotechniques 13: 852-854 (1992)); reverse is intended to still more clearly distinguish between LSCs and transcription polymerase chain reaction (RT-PCR) (Weis et HSCs, it is preferable that the following marker genes (3), out al., Trends in Genetics 8: 263-264 (1992)); realtime quanti of the marker genes (2), be used as an index. The marker tative RT-PCR (Held et al., Genome Research 6: 986-994 genes (3) are more preferable because normally 5 times or (1996)); microarray analysis and the like. Microarray analy higher differential expression is observed in LSCs than in sis can be performed using, the Affymetrix GeneChip tech HSCs. In this mode of embodiment, the marker genes (3) nique, the microarray technique of Agilent Technologies or consist of 2 to 35 genes, more preferably consist of 3 or more, the microarray technique of Incyte with a commercially avail 5 or more, 10 or more, 15 or more, 20 or more, or 25 or more, able apparatus, as directed by the manufacturer. Details of genes. realtime quantitative RT-PCR are described in Examples 0038 Marker Genes (2): below. Examples of the base sequences of primers and probes cell membrane- or extracellularly-localized genes consisting that are suitably used for realtime quantitative RT-PCR are of ADFP. ALOX5AP CACNB4, CCL5, CD33, CD3D, listed in Table 3 and the sequence listing. CD93, CD97, CLEC12A, DOK2, FCER1G, FCGR2A, 0044) When the translation product of a marker gene is the FUCA2, GPR34, GPR84, HCST, HGF, HOMER3, IL2RA, analyte, protein can be isolated from the biological sample IL2RG, IL3RA, ITGB2, LGALS1, LRG1, LY86, MGAT4A, according to a conventional method. Ordinary methods for P2RY5, PRSS21, PTH2R, RNASE2, SLC43A3, SUCNR1, protein extraction are well known in the relevant technical TIMP1, TNF, TNFRSF4, TNFSF13B, TYROBP and VNN1; field, and are disclosed in standard textbooks of molecular cell cycle-related genes consisting of ZWINT, NEK6 and biology, including Ausubel et al., Current Protocols of TXNL4B; an apoptosis-related gene consisting of BIK; sig Molecular Biology, John Wiley and Sons (1997) and the like. naling-related genes consisting of AK5, ARHGAP18. FYB, Isolation of protein can be achieved using purification kits, HCK, LPXN, PDE9A, PDK1, PRKCD, RAB20, RAB8A and buffer solution sets, and protease inhibitors obtained from RABIF; transcription factor genes consisting of WT1 and their manufacturers, as directed by the manufacturers. HLX; and other genes consisting of CYBB, CTSC and NCF4. 0045. The method of measuring the expression level of a 0039 Marker Genes (3): marker gene for a translation product as an analyte is not cell membrane- or extracellularly-localized genes consisting particularly limited; available methods include the immuno of ALOX5AP CACNB4, CCL5, CD33, CD3D, CD93, histochemical method, the proteomics method and the like. CD97, CLEC12A, DOK2, FCGR2A, GPR84, HCST, The immunohistochemical method comprises detecting the HOMER3, ITGB2, LGALS1, LRG1, PTH2R, RNASE2, expression using an antibody specific for each marker gene TNF, TNFSF13B, TYROBP and VNN1; a cell cycle-related product. Protocols and kits for the immunohistochemical gene consisting of NEK6; an apoptosis-related gene consist method are well known in the relevant technical field, and are ing of BIK; signaling-related genes consisting of AK5, FYB, commercially available. The proteomics method comprises HCK, LPXN, PDE9A, PDK1, PRKCD and RAB20; a tran examining overall changes in protein expression in a certain Scription factor gene consisting of WT1; and other genes sample. The proteomics method generally comprises the fol consisting of CTSC and NCF4. lowing steps: (1) separation of various in the sample 0040 Although the subject in the test method of the by 2-D gel electrophoresis (2-D PAGE), (2) identification of present invention is not particularly limited, as far as it is a the various proteins recovered from this gel by, for example, mammal, including a human, a human Suspected of Suffering mass analysis or N-terminal sequencing, and (3) data analysis the initial onset or a recurrence of leukemia is preferred. using bioinformatics. The proteomics method is a useful 0041. The biological sample to be measured by the test method for Supplementing other gene expression profiling method of the present invention is not particularly limited, as methods, and can be used alone, or in combination with far as it can be collected from a mammal, preferably from a another method, to detect products of marker genes of the human; examples include humoral samples Such as blood, present invention. When a cell Surface marker is the target, bone marrow fluid, and lymph fluid, and solid samples Such as measuring method using flow cytometry is possible. lymph nodes, blood vessels, bone marrow, brain, spleen, and (2) Step of comparing the expression levels obtained in the skin. measuring step with a reference value 0042. In the test method of the present invention, the 0046 When the results of measurements of the expression expression level of a marker gene is measured for a transcrip levels of2 to 218 kinds of marker genes in a biological sample tion product or translation product of the gene as an analyte. show that the expression levels of 2 kinds or more thereofare When the transcription product is the analyte, RNA can be significantly higher than reference values (gene expression isolated from the biological sample by a conventional differs about 2 fold or more, preferably about 4 fold or more, method. Ordinary methods for RNA extraction are well more preferably about 6 fold or more, most preferably about known in the relevant technical field, and are disclosed in 10 fold or more), the possible presence of a leukemic stem cell standard textbooks of molecular biology, including Ausubel in the sample or the Subject's body is suggested. Here, useful et al., Current Protocols of Molecular Biology, John Wiley reference values include comparator values such as mean and Sons (1997) and the like. Specifically, isolation of RNA expression levels for healthy persons and mean levels for the can be achieved using purification kits, buffer solution sets, Subject before onset. The Suggestion of the possible presence US 2012/0070450 A1 Mar. 22, 2012

of leukemic stem cell leads to prediction of the initial onset or ingredients may be contained in a single pharmaceutical a recurrence of leukemia in the subject. It is preferable that the preparation, or may be contained in separate pharmaceutical presence or absence of the initial onset or a recurrence of preparations. leukemia be checked by another test. 0051 Described below are active ingredients. 0047. In the test method of the present invention, when the results of measurements of the expression levels of 2 to 218 0.052 Substances capable of suppressing the expression of kinds of marker genes in a biological sample show that the a leukemic stem cell marker gene include, for example, anti expression levels of 2 kinds or more thereof are significantly sense nucleic acids, RNAi-inducible nucleic acids and the higher than reference values (gene expression differs about 2 like. fold or more, preferably about 4 fold or more, more preferably 0053 Substances capable of suppressing the activity of a about 6 fold or more, most preferably about 10 fold or more), translation product of a leukemic stem cell marker gene the possible presence of a leukemic stem cell in the sample or include, for example, aptamers, antibodies and the like. The the body of the source from which the sample has been Substance may be an inhibitory Substance that acts directly or collected is suggested. Here, useful reference values include indirectly on each marker. comparator values such as mean expression levels for healthy persons and mean expression level for the subject before 0054 Described below are active ingredients of the thera onset. In this case, the Suggestion of the possible presence of peutic agent of the present invention. a leukemic stem cell leads to prediction that the treatment is not completely effective on the cancer in the leukemiapatient. 1. Antisense Nucleic Acid Conversely, when the expression levels of the aforemen tioned 2 kinds or more are significantly lower (for example, 0055. The kind of the antisense nucleic acid may be DNA substantially Zero), it can be predicted that leukemic stem or RNA, or a DNA/RNA chimera. The antisense nucleic acid cells are absent in the sample. In this case, it is thought that the may be one having a natural type phosphoric acid diester treatment of leukemia eliminated leukemic stem cells and is bond, or a modified nucleotide such as of the thiophosphate effective. Furthermore, it is preferable that the test method be type (P=O in phosphate linkage replaced with P=S), 2'-O- combined with other examinations to achieve multi-angle methyl type and the like, which are stable to decomposing confirmation of a therapeutic effect on leukemia. enzymes. Other important factors for the designing of anti 0048. As stated above, by applying the test method of the sense nucleic acids include increases in water-solubility and present invention, it is possible to detect leukemic stem cells cell membrane permeability and the like; these can also be in a living organism before leukemia occurs initially or cleared by choosing appropriate dosage forms such as those recurs, and predict the onset. Alternatively, it is also possible using liposome or microspheres. The length of the antisense to detect the onset of leukemia in the initial stage and lead to nucleic acid is not particularly limited, as far as the antisense early treatment of cancer patients. Furthermore, it is also nucleic acid is capable of specifically hybridizing with the possible to evaluate the therapeutic effect on leukemia transcription product; the antisense nucleic acid may be a patients with the presence or absence of leukemic stem cells sequence comprising about 15 nucleotides for the shortest, or as an index. comprising a sequence complementary to the entire sequence of the transcription product for the longest. Taking into (Therapeutic Agent) account the issues of the ease of synthesis, antigenicity and 0049. The present invention also provides a therapeutic the like, oligonucleotides consisting of for example, about 15 agent for acute myeloid leukemia that targets leukemic stem or more nucleotides, preferably about 15 to about 100 nucle cells, comprising as an active ingredient a Substance capable otides, more preferably about 18 to about 50 nucleotides, can of suppressing the expression of a leukemic stem cell marker be mentioned as examples. Furthermore, the antisense gene or a Substance capable of Suppressing the activity of a nucleic acid may be one that not only hybridizes with the translation product of the gene. transcription product to inhibit the translation, but also is 0050 Molecular targets for the therapeutic agent of the capable of binding to a double-stranded DNA to form a triple present invention are the above-described leukemic stem cell strand (triplex) to inhibit the transcription into mRNA. marker genes, and any marker may be selected according to the purpose of treatment. When the therapeutic agent of the 2. RNAi-Inducible Nucleic Acid present invention targets stem cells, out of leukemic stem cells, that are present in bone marrow niches, are in the 0056. An RNAi-inducible nucleic acid refers to a poly stationary phase of cell cycle, and are resistant to anticancer nucleotide, preferably an RNA, capable of inducing the RNA agents, it is recommended that a Substance capable of Sup interference (RNAi) effect when introduced into cells. The pressing the expression of genes selected from the group RNAi effect refers to the phenomenon in which a double consisting of AK5, BIK, DOK2, FCGR2A, IL2RA, LRG1, Stranded RNA comprising the same nucleic acid sequence as SUCNR1 and WT1 (hereinafter also referred to as marker that of mRNA, or a partial sequence thereof. Suppresses the genes (4)) or a Substance capable of Suppressing the activity expression of the mRNA. To obtain the RNAi effect, it is of a translation product of the gene be selected. By selecting preferable to use, for example, a double-stranded RNA hav 2 to 8 (preferably 2 to 5) out of the eight genes constituting the ing the same nucleic acid sequence as that of the target mRNA marker genes (4) and using them as molecular targets, it is comprising at least 19 continuous bases (or a partial sequence highly likely possible to exterminate leukemic stem cells of a thereof). The double-stranded structure may be configured by large number of patients. Therefore, at least one active ingre different strands, or may be a double strand conferred by a dient is contained in the therapeutic agent of the present stem-loop structure of one RNA. Examples of RNAi-induc invention, and it is preferable that two or more be combined ing nucleic acids include siRNAs, miRNAs and the like, with according to the purpose of treatment. Two or more active preference given to siRNAs. The siRNA is not particularly US 2012/0070450 A1 Mar. 22, 2012

limited, as far as it can induce an RNAi, and the siRNA can be, days after final administration, the spleen or lymph nodes are for example, 19 to 27 bases long, preferably 21 to 25 bases collected, and leukocytes are collected. These leukocytes and long. myeloma cells (for example, NS-1, P3X63Ag8 and the like) are cell-fused to obtain a hybridoma that produces a mono 3. Aptamer clonal antibody against the factor. This cell fusion may be performed by the PEG method or the voltage pulse method. A 0057. An aptamer refers to a polynucleotide having a hybridoma that produces the desired monoclonal antibody binding activity (or inhibitory activity) on a specified target can be selected by detecting an antibody that binds specifi molecule. An aptamer is an RNA, a DNA, a modified nucle cally to the antigen in the culture Supernatant, using a widely otide or a mixture thereof. The aptamer can be in a linear or known EIA or RIA method and the like. Cultivation of the circular form. The length of the aptamer is not particularly hybridoma that produces the monoclonal antibody can be limited, and is normally about 16 to about 200 nucleotides: performed in vitro, or in vivo such as in ascitic fluid of a for example, the length is about 100 nucleotides or less, mouse or rat, preferably a mouse, and the antibody can be preferably about 50 nucleotides or less, and more preferably acquired from the culture supernatant of the hybridoma or the about 40 nucleotides or less. The length of the aptamer may ascitic fluid of the animal. be, for example, about 18, about 20, about 25 or about 30, nucleotides or more. The aptamer, for increasing the bind 0061 The antibody may be a chimericantibody, a human ability, stability, drug delivering quality and the like, may be ized antibody or a human antibody. one wherein a Sugar residue (e.g., ribose) of each nucleotide 0062. A chimericantibody means a monoclonal antibody is modified. Examples of portions of the sugar residue where derived from immunoglobulins of animal species having it is modified include ones wherein the oxygen atom at the mutually different variable regions and constant regions. For 2'-position, 3'-position and/or 4'-position of the Sugar residue example, the chimeric antibody can be a mouse/human chi is replaced with another atom and the like. Examples of types meric monoclonal antibody whose variable region is a vari of modifications include fluorination, O-alkylation, O-allyla able region derived from a mouse immunoglobulin, and tion, S-alkylation, S-allylation and amination (see, e.g., whose constant region is a constant region derived from a Sproat et al., (1991) Nucle. Acid. Res. 19, 733–738; Cotton et human immunoglobulin. The constant region derived from a al., (1991) Nucl. Acid. Res. 19, 2629-2635). Theaptamer may human immunoglobulin has an amino acid sequence unique also be one wherein a purine or pyrimidine is altered. depending on the isotype, IgG, IgM, IgA, Ig|D, IgE or the like, Examples of such alterations include alteration of the 5-po and the constant region of a recombinant chimeric mono sition pyrimidine, alteration of the 8-position purine, alter clonal antibody in the present invention may be the constant ation with an exocyclic amine, Substitution with 4-thiouri region of a human immunoglobulin belonging to any isotype. dine, and substitution with 5-bromo or 5-iodo-uracil. The The constant region of human IgG is preferable. phosphate group contained in the aptamer of the present 0063 A chimeric antibody can be prepared by a method invention may be altered to make it resistant to nucleases and known perse. For example, a mouse/human chimeric mono hydrolysis. For example, the phosphate group may be substi clonal antibody can be prepared according to available tuted with a thioate, a dithioate oran amidate. Anaptamer can reports (e.g., Jikken Igaku (extra issue), Vol. 6, No. 10, 1988 be prepared according to available reports (for example, and JP-B-HEI-3-73280). In detail, a mouse/human chimeric Ellington et al., (1990) Nature, 346, 818-822; Tuerk et al., monoclonal antibody can be prepared by inserting the C. (1990) Science, 249,505-510). gene acquired from the DNA that encodes a human immuno globulin (C gene that encodes H chain constant region) down 4. Antibody stream of the active V gene acquired from the DNA that encodes a mouse monoclonal antibody, isolated from a hybri 0058. The antibody may be a polyclonal antibody (antise doma that produces the mouse monoclonal antibody (rear rum) or a monoclonal antibody, and can be prepared by a ranged VDJ gene that encodes H chain variable region), and commonly known immunological technique. Although the inserting the C gene acquired from the DNA that encodes a monoclonal antibody may be of any isotype, IgG, IgM, IgA, human immunoglobulin (C gene that encodes L chain con Igl), IgE, or the like, IgG or IgM is preferable. stant region) downstream of the active V, gene acquired from 0059 For example, the polyclonal antibody can be the DNA that encodes amouse monoclonal antibody, isolated acquired by Subcutaneously or intraperitoneally administer from the hybridoma (rearranged VJ gene that encodes L chain ing the above-described antigen (as required, may be pre variable region), into one or separate expression vectors in a pared as a complex crosslinked to a carrier protein Such as way that allows the expression of each gene, transforming a bovine serum albumin or KLH (Keyhole Limpet Hemocya host cell with the expression vector, and culturing the trans nin)), along with a commercially available adjuvant (for formant cell. example, Freund's complete or incomplete adjuvant), to an 0064. A humanized antibody means a monoclonal anti animal about 2 to 4 times at intervals of 2 to 3 weeks (the body prepared by a gene engineering technique, for example, antibody titer of partially drawn serum has been determined a human type monoclonal antibody wherein some orall of the by a known antigen-antibody reaction and its elevation has complementarity-determining regions of the ultra-variable been confirmed in advance), collecting whole blood about 3 region thereof are derived from a mouse monoclonal anti to 10 days after final immunization, and purifying the antise body, and the framework region of the variable region thereof rum. Animals to receive the antigen include mammals such as and the constant region thereof are derived from a human rats, mice, rabbits, goat, guinea pigs, and hamsters. immunoglobulin. The complementarity-determining regions 0060. The monoclonal antibody can also be prepared by of the ultra-variable region are three regions that are present in cell fusion. For example, the above-described antigen, along the ultra-variable region in the variable region of the antibody, with a commercially available adjuvant, is subcutaneously or and that complementarily bind directly to the antigen intraperitoneally administered to a mouse 2 to 4 times, and 3 (complementarity-determining regions; CDR1, CDR2, US 2012/0070450 A1 Mar. 22, 2012

CDR3), and the framework regions of the variable region are 0069. The above-described antibody may be in the form of four relatively highly conserved regions interposing the front an immunoconjugate bound with various anticancer Sub and back of the three complementarity-determining regions stances and the like by a conventional method. In this case, the (frameworks; FR1, FR2, FR3, FR4). Hence, a humanized antibody functions as a drug delivery system for delivering an antibody means, for example, a monoclonal antibody anticancer agent to LSCs. Anticancer Substances to be com wherein all regions other than Some or all of the complemen bined include, but are not limited to, cisplatin, carboplatin, tarity-determining regions of the ultra-variable region of a cyclophosphamide, melphalan, carmusulin, methotrexate, mouse monoclonal antibody are replaced with corresponding 5-fluorouracil, cytarabine (AraC), mercaptopurine, daunoru regions of a human immunoglobulin. bicin, idarubicin, mitoxantrone, thioguanine, azacitidine, 0065. A humanized antibody can be prepared by a method amsacrine, doxorubicin, tretinoin, allopurinol, prednisone known per se. For example, a recombinant humanized anti (prednisolone), epirubicin, vinblastine, Vincristine, dactino body derived from a mouse monoclonal antibody can be mycin (actinomycin), mitomycin C, taxol. L-asparaginase, prepared according to available reports (e.g., Japanese Patent etoposide, colchicine, deferoxamine mesylate, camptothecin Application Kohyo Publication No. HEI-4-5064.58 and JP-A- and the like. Furthermore, the antibody may be an immuno SHO-62-296890). In detail, from a hybridoma that produces conjugate with a radionuclide, toxin and the like. a mouse monoclonal antibody, at least one mouse H chain 0070 The agent of the present invention can comprise, in CDR gene and at least one mouse L chain CDR gene corre addition to a Substance capable of suppressing the expression sponding to the mouse H chain CDR gene are isolated, and of a leukemic stem cell marker gene or the activity of a from a human immunoglobulingene, the human H chain gene translation product of the gene, an optionally chosen carrier, that encodes all regions other than the human H chain CDR for example, a pharmaceutically acceptable carrier. Examples corresponding to the mouse H chain CDR and the human L of pharmaceutically acceptable carriers include, but are not chain gene that encodes all regions other than the human L limited to, excipients such as Sucrose, starch, mannitol, Sor chain CDR corresponding to the mouse L chain CDR are bitol, lactose, glucose, cellulose, talc, calcium phosphate and isolated. The mouse H chain CDR gene and human H chain calcium carbonate; binders such as cellulose, methylcellu gene isolated are introduced into an appropriate expression lose, hydroxypropylcellulose, polypropylpyrrolidone, gela vector expressibly; likewise, the mouse L chain CDR gene and the human L chain gene are introduced into another tin, acacia, polyethylene glycol, Sucrose and starch; disinte appropriate expression vector expressively. Alternatively, the grants such as Starch, carboxymethylcellulose, mouse H chain CDR gene/human H chain gene and the mouse hydroxypropyl starch, sodium-glycol-starch, sodium hydro L chain CDR gene/human L chain gene can be introduced gen carbonate, calcium phosphate and calcium citrate; lubri into the same expression vector expressively. By transform cants such as magnesium Stearate, Aerosil, talc and Sodium ing a host cell with the expression vector thus prepared, it is lauryl Sulfate; flavoring agents such as citric acid, menthol, possible to obtain a cell that produces a humanized antibody, glycyrrhizin ammonium salt, glycine and orange powder; and by culturing the cell, the desired humanized antibody can preservatives such as Sodium benzoate, sodium hydrogen be obtained from the culture supernatant. Sulfite, methyl paraben and propyl paraben; stabilizers such 0066. A human antibody means an antibody wherein all as citric acid, sodium citrate and acetic acid; Suspending regions comprising the variable regions and constant regions agents such as methylcellulose, polyvinylpyrrolidone and of the H chain and L chain constituting an immunoglobulin aluminum Stearate; dispersing agents such as Surfactants; are derived from the gene that encodes a human immunoglo diluents such as water, physiological Saline and orange juice; bulin. base waxes such as cacao butter, polyethylene glycol and 0067. A human antibody can be prepared by a method refined kerosene; and the like. known per se. For example, a human antibody can be pro 0071 Preparations suitable for oral administration are liq duced by immunologically sensitizing with an antigen a uids prepared by dissolving an effective amount of a Sub transgenic animal prepared by incorporating at least a human stance in a diluent Such as water or physiological saline, immunoglobulin gene into a gene locus of a non-human capsules, Sachets or tablets containing an effective amount of mammal Such as a mouse, in the same way as the above a Substance in the form of Solids or granules, Suspensions described method of preparing a polyclonal antibody or a prepared by Suspending an effective amount of a Substance in monoclonal antibody. For example, a transgenic mouse that an appropriate dispersant, emulsions prepared by dispersing produces a human antibody can be prepared according to and emulsifying a solution of an effective amount of a Sub available reports (Nature Genetics, Vol. 15, p. 146-156, 1997: stance in an appropriate dispersant, or powders, granules and Nature Genetics, Vol. 7, p. 13-21, 1994; Japanese Patent the like. Application Kohyo Publication No. HEI-4-504365: Interna 0072 Preparations suitable for parenteral administration tional Patent Application Publication WO94/25585; Nature, (e.g., intravenous injection, Subcutaneous injection, intra Vol. 368, p. 856-859, 1994; and Japanese Patent Application muscular injection, topical injection and the like) are aqueous Kohyo Publication No. HEI-6-500233). and non-aqueous isotonic sterile injectable liquids, which 0068. The antibody may be a part of the above-mentioned may contain an antioxidant, a buffer Solution, a bacteriostatic antibody (e.g., monoclonal antibody). The antibody may be a agent, an isotonizing agent and the like. Aqueous and non fragment such as F(ab'), Fab', Fab, Fv and the like, a conju aqueous sterile Suspensions can also be mentioned, which gate molecule prepared by genetic engineering Such as Schv, may contain a suspending agent, a solubilizer, a thickening schv-Fc, minibody, diabody and the like, or a derivative agent, a stabilizer, an antiseptic and the like. These prepara thereof, which is modified by a molecule and the like having tions can be enclosed in containers such as ampoules and vials a proteinstabilizing action Such as polyethylene glycol (PEG) for unit dosage or a plurality of dosages. It is also possible to and the like, and the like. freeze-dry the active ingredient and a pharmaceutically US 2012/0070450 A1 Mar. 22, 2012 17 acceptable carrier, and store the preparation in a state that may (Phosphate Buffered Saline). After the washing step is be dissolved or Suspended in an appropriate sterile vehicle repeated several times, a sample containing hematopoietic just before use. cells can be obtained. 0073. Although the dosage of the agent of the present 0078. In the case of peripheral blood, collection is per invention varies depending on the activity and choice of formed from a vein. Specifically, the skin surface of the active ingredient, the mode of administration (e.g., oral, portion for peripheral blood collection is disinfected. The parenteral), the seriousness of disease, the animal species inner cylinder of the injection needle is removed, a 10 mL which is the Subject of administration, the drug tolerance, Syringe containing 5000 units of heparin is attached, and the body weight and age of the Subject of administration, and the required amount of peripheral blood is quickly aspirated. On like, and cannot be generalized, it is normally about 0.001 mg average, 10 mL to 20 mL of peripheral blood is aspirated. The to about 5.0 gas the amount of active ingredient per day for an injection needle is removed, and astriction is performed for adult. about 10 minutes. The peripheral blood acquired is centri 0074 The subject of administration of the agent of the fuged at 1,000xg, and peripheral blood cells are recovered, present invention is not particularly limited, as far as it is an after which the peripheral blood cells are washed with PBS animal species having a hematopoietic tissue (bone marrow), (Phosphate Buffered Saline). After the washing step is and possibly contracting acute myeloid leukemia, and it is repeated several times, a sample containing hematopoietic preferably a mammal, more preferably a human. cells can be obtained. 0079 b) Step of bringing the collected sample into contact (Method of Production) with a Substance that recognizes a translation product of at 0075. The present invention also provides a method for least one kind of leukemic stem cell marker gene producing a sample containing hematopoietic cells for 0080. The substances that recognize a translation product autologous transplantation or allogeneic transplantation for a of the marker genes for use in this step include antibodies patient with acute myeloid leukemia. The production method described above, with particular preference given to antibod of the present invention comprises, ies against at least one kind of cell Surface marker selected a) a step of collecting a sample containing hematopoietic cells from among ADFP. ALOX5AP, CACNB4, CD33, CD3D, from the patient or a donor, CD93, CD97, CLEC12A, DOK2, FCER1G, FCGR2A, b) a step of bringing the collected sample into contact with at GPR34, GPR84, HCST, HOMER3, IL2RA, IL2RG, IL3RA, least one kind of substance that recognizes a translation prod ITGB2, LY86, P2RY5, PTH2R, SUCNR1, TNFRSF4, uct of a leukemic stem cell marker gene, and TYROBP and VNN1. Preferably, the antibodies are fluores c) a step of sorting cells to which the above-described sub cently labeled, and preferable fluorescent dyes used for the stance has been bound, and obtaining the sample from which labeling are fluorescent Substances commonly used for flow leukemic stem cells have been purged. Accordingly, the cytometry. Specific examples of fluorescent dyes include present invention makes it possible to Substantially remove FITC (fluorescein isothiocyanate), PE (phycoerythrin), leukemic stem cells from a sample containing hematopoietic PerCP (peridinin-chlorophyll-protein), PerCP-Cy5.5, cells for autologous transplantation or allogeneic transplan PE-Cy5, PE-Cy7, PE-TR (PE-Texas Red), APC (allophyco tation, and provide a sample for transplantation without the cyanin), APC-Cy7 and the like. Conditions for the contacting fear of recurrences. are not particularly limited, as far as a contact between the 0076. The leukemic stem cell marker genes are as men above-mentioned cell Surface marker (antigen) and the anti tioned above; for the purpose of purging, however, it is pre body can be achieved. ferred to target at least one kind of cell Surface marker gene I0081 c) Step of sorting cells to which the above-described selected from among the following set of genes: Substance has bound, and obtaining the sample from which ADFP. ALOX5AP, CACNB4, CD33, CD3D, CD93, CD97, leukemic stem cells have been purged CLEC12A, DOK2, FCER1G, FCGR2A, GPR34, GPR84, I0082 In this step, cell sorting can easily be accomplished HCST, HOMER3, IL2RA, IL2RG, IL3RA, ITGB2, LY86, by combining with flow cytometry. The sample in contact P2RY5, PTH2R, SUCNR1, TNFRSF4, TYROBP and with a fluorescently labeled antibody is set to a flow cytom VNN1. eter, and the cells bound to the antibody are sorted; leukemic a) Step of collecting a sample containing hematopoietic cells stem cells can be separated from the sample. from a patient with acute myeloid leukemia or a donor I0083. The thus-obtained LSC-purged sample can be used 0077 Sample collection is normally achieved by bone for the treatment of AML patients, without the fear of recur marrow aspiration or peripheral blood collection. Bone mar rences, as the LSCs have been efficiently eliminated, whereas row aspiration is performed on the sternum or ilium on the HSCs have been concentrated escaping elimination. basis of, for example, the method described in S. E. Haynes worth et al., Bone, 13, 81 (1992) and the like. Specifically, the EXAMPLES skin Surface of the portion for aspirating the bone marrow is disinfected, and topical anesthesia is performed. The Subpe I0084. The present invention is hereinafter described in riosteal region, in particular, is anesthetized Sufficiently. The detail by means of the following Examples, by which, how inner cylinder of the puncture needle is removed, a 10 mL ever, the invention is not limited in any way. Syringe containing 5000 units of heparin is attached, and the required amount of bone marrow fluid is quickly aspirated. Human Samples On average, 10 mL to 20 mL of bone marrow fluid is aspi I0085 All experiments were conducted with the approval rated. The puncture needle is removed, and astriction is per of the Institutional Review Board for Human Research of the formed for about 10 minutes. The bone marrow fluid acquired RIKEN Research Center for Allergy and Immunology. Leu is centrifuged at 1,000xg, and bone marrow cells are recov kemia cells derived from AML patients were collected with ered, after which the bone marrow cells are washed with PBS informed consent in writing. CB (cord blood) derived from US 2012/0070450 A1 Mar. 22, 2012 healthy donors, along with informed consent in writing, was Quantitative PCR (qPCR) Analysis collected by the Tokyo Cord Blood Bank. BMMNCs (bone I0088 Ten ng of total RNA from HSCs or LSCs was sub marrow mononuclear cells) derived from healthy donors jected to cDNA amplification using WTOvation RNA were obtained from Cambrex (Walkerville, Md.). BMMNCs Amplification System (Nugen). The cDNA products were and CBMNCs (cord blood mononuclear cells) derived from diluted 1:7.5 in TE, and 1 ul of the dilution products was used AML patients were isolated using density gradient centrifu per 25ul of qPCR reaction. The sequences of doubly-labeled gation. fluorescent probes and gene specific primers (Sigma-Aldrich) were listed in Table 3. PCR reactions were performed using FACS and Flow Cytometric Analysis LightCycler 480 (Roche Applied Science) with Platinum I0086 For fluorescence-activated cell sorting (FACS), Quantitative PCR SuperMix-UDG (Invitrogen). The abun BMMNC cells from AML patients were labeled with fluo dance of the respective transcripts was calculated by the stan rescent dye-coupled mouse anti-hCD3, anti-hCD4, anti dard curve method (Methods, 25, 386-401, 2001). When any hCD8, anti-hCD34 and anti-hCD38 monoclonal antibodies of Kruskal-Wallis, Wilcoxon-Mann-Whitney and Student's (BD Biosciences, San Jose, Calif.), and recipient BMMNC t-test in Kaleida Graph software package showed P-0.05, it cells were labeled with mouse anti-hCD45, anti-hCD34 and was determined there is a significant difference in the expres anti-hCD38 monoclonal antibodies (BD Biosciences); the Sion level between LSC and HSC. cells were sorted using FACSAria (BD Biosciences). Dou blets were eliminated via analyzing FSC/SSC height and Animals FSC/SSC width. After the sorting, the purity of hCD34+ hCD38- and hCD34+ cells was higher than 98%. For flow I0089 NOD.Cg-Prkdc'I12rg"/Sz (NOD/SCID/ cytometric analysis, BMMNCs of AML patients, recipient IL2rgy") mice were developed at The Jackson Laboratory peripheral blood or recipient BM was labeled with the above (Bar Harbor, Me.) by backcrossing a complete null mutation described fluorescent dye-coupled mouse anti-hCD3, anti at the I12rg locus onto the NOD.Cg-Prkdc' (NOD/SCID) hCD4, anti-hCD8, anti-hCD34 and anti-hCD38 monoclonal strain (Shultz, L. D. et al. Multiple defects in innate and antibodies or mouse anti-hCD45, anti-hCD34 and anti adaptive immunologic function in NOD/LtSz-scid mice. J hCD38 monoclonal antibodies. Immunol 154, 180-191 (1995)). Mice were bred and main tained under defined flora with irradiated food and acidified Microarray Analysis water at the animal facility of RIKEN and at The Jackson 0087 Total RNA was extracted using TRIZol Reagent (In Laboratory according to guidelines established by the Insti vitrogen), and the integrity of the RNA was then assessed tutional Animal Committees at the respective institutions. with Agilent Bioanalyzer. Biotinylated cRNAs were synthe sized using Two-Cycle Target Labeling Kit (Affymetrix) for Heterologous Transplantation U133 plus 2.0 GeneChip (Affymetrix). For Human Gene 1.0ST GeneChip (Affymetrix), a first round of (0090 Newborn (within 2 days of birth) NOD/SCID/ cDNA synthesis and crNA amplification were performed IL2rg" mice received 150 cGy of total body irradiation using Message Amp Premier RNA Amplification Kit (Ap using a '7Cs-source irradiator, followed by intravenous plied Biosystems), and a subsequent second round of cDNA injection of AML cells within two hours. The recipients were synthesis, biotinylation and fragmentation were performed subjected to blood sampling from retro-orbital every 3-4 using WT cDNA Synthesis and Terminal Labeling kits (Af weeks, and human AML transplantation chimerism in periph fymetrix). Hybridization, washing, staining and scanning eral blood was assessed. were performed according to the manufacturers instruction. Firstly, the microarray data for each platform was separately Immunofluorescent Labeling and Imaging analyzed using Bioconductor package (http://www.biocon 0091 Para-formaldehyde-fixed decalcified paraffin-em ductor.org/). The signal intensities of probe sets on the bedded sections were prepared from a femoral bone of a microarray platforms were normalized with GC-RMA pro primary AML transplantation recipient. The primary antibod gram (Zhijin et al., J. Am. Stat. Assoc., 99,909-917, 2004). ies used for labeling were a mouse anti-human CD45 mono For each platform, the normalized data was analyzed with clonal antibody (DAKO, Denmark) and a rabbit anti-CD32 RankProd program (Hong et al., Bioinformatics, 22, 2825 monoclonal antibody (Abcam, UK). Laser Scanning confocal 2827, 2006) to select genes differentially expressed between imaging was obtained using Zeiss LSM Exciterand LSM 710 LSCs and HSCs with the cutoff p value of 0.01 and the (Carl Zeiss). false-positive estimation of 0.05%. When a significantly higher level of expression was observed in LSC than in HSC Immunofluorescent Labeling and Imaging (2) commonly in both the microarray platforms, the gene was selected as a significant candidate LSC marker gene (FIG. 5, 0092 Para-formaldehyde-fixed decalcified paraffin-em Table 1). In addition, the gene IL2RA, which gave a high hit bedded sections were prepared from a femoral bone of a rate for Human Gene 1.0ST GeneChip, and provided favor recipient of transplantation of primary AML treated with an able results in the protein level analysis, was also selected as anticancer agent, and stained with antibodies against DAPI a candidate marker gene, since it is expressed in stem cells (nuclear staining: blue); various markers (FCGR2A, AK5, resistant to anticancer drugs as described below (Table 1). DOK2, LRG1, BIK, IL2RA, WT1, SUCNR1: red); and sta The localization and the biological function of the candidates tionary cell markers (green: CD34 (FCGR2A, AK5, DOK2, were annotated based on information from Ingenuity Path LRG1, BIK) or Ki67 (IL2RA, WT1, SUCNR1). Laser scan way Analysis Database (Ingenuity Systems) and Gene Ontol ning confocal imaging was obtained using Zeiss LSMEXciter ogy Annotation Database (http://www.ebi.ac.uk/GOA?). and LSM 710 (Carl Zeiss) (FIG. 7).

US 2012/0070450 A1 Mar. 22, 2012 26

TABLE 3 - continued List of primers, probes and PCR products used in qRT-PCR s RAB20 GCCTTCTACCTGAAGCAGTGGCGGCTGGTACAACATCTCCATCTGGGACACCGCAGGGC 14 O GGGAGCAGTTCCAACGGCCTGGGTCCATGTACTGCCGGGGGGCGGGGGGGATCATCCT CACCTAGATGTGAATCACCGGCA (SEO ID NO: 218) s RAB8A GCCAACATCAATGTGGAAAATGGATTTTTCAGTGTCGCCAGAGATATCAAAGCAAAA 14 O TGGACAAAAAATTGGAAGGCAACAGCCCCCAGGGGAGCAACCAGGGAGTCAAAATCAC ACCGGACCAGCAGAAGAGGAGCAG (SEQ ID NO: 219) s RABIF AGGGACCCCTCTCTTCTCTCGCCCACAGCTTTTCCTTCCCTCCATGAGAAAGAAGCCA 119 GCTCTGTCTGACGGGAGCAATCCTGAGGGCCATCTCCTGCAGGAACACTGGGTGGTTG AGG (SEQ ID NO: 220) s RNSE2 CCCTGAACCCCAGAACAACCAGCTGGATCAGTTCTCACAGGAGCTACAGCGCGGAGAC 91 TGGGAAACATGGTTCCAAAACTGTTCACTTCCC (SEO ID NO: 221) s SLC43A AAGCTCTTTGGGCTGGTGATGGCCTTGTCGGCTCTGGTGTCTCTGCTCCAGTTCCCA 148 TCTTCACCCTCATCAAAGGCTCCCTTCAGAATGACCCATTTTACGTGAATGTGAGTT CATGCTTGCCATTCTTCTGACATTCTTCCACC (SEQ ID NO: 222) s SUCNR1 TACGACATGCTGGGGATCATGGCATGGAATGCAACTTGCAAAAACTGGCTGGCAGCAG 150 AGGCTGCCCTGGAAAATACTACCTTTCCATTTTTTATGGGATTGAGTTCGTTGTGGG AGTCCTTGGAAATACCATTGTTGTTTACGGCTAC (SEO ID NO ; 223) s TIMP1 ACTGTTGGCTGTGAGGAATGCACAGTGTTTCCCTGTTTATCCATCCCCTGCAAACTGC 112 AGAGTGGCACTGATTGCTTGTCCACGGACCAGCTCCTCCAAGGCTCTGAAAAGG (SEQ ID NO: 224) s TNF CCAGGCAGTCAGATCATCTTCTCGAACCCCGAGTGACAAGCCTGTAGCCCATGTTGTA 14 O GCAAACCCTCAAGCTGAGGGGCAGCTCCAGTGGCTGAACCGCCGGGCCAATGCCCTCG TGGCCAATGGCGTGGAGCTGAGAG (SEQ ID NO: 225) s TNFRSF(2) CCTGCAAGCCCTGGACCAACTGCACCTTGGCTGGGAAGCACACCCTGCAGCCGGCCAG 78 CAATAGCTCGGACGAATCT (SEO ID NO : 226) (2s TNFSF13(2) ACCAGCTCCAGGAGAAGGCAACTCCAGTCAGAACAGCAGAAATAAGCGTGCCGTTCAG 97 GGTCCAGAAGAAACAGTCACTCAAGACTGCTTGCAACTG (SEO ID NO: 227

TCCGAGAAGTGGTTGCTGACAGCCACAAAGTGAAAGGGAGTGAGGCGGCGTGGACGAG 1 OO TAAGGAGTGACAGTGAGGATTCACATTTGGGTTATTTCAAGA (SEO ID NO : 228) Hs. TYRO8P CTGAGACCGAGTCGCCTTATCAGGAGCTCCAGGGTCAGAGGTCGGATGTCTACAGCGA 8O CCTCAACACACAGAGGCCGTAT (SEO ID NO: 229) His VNN1 AGTGCTGTGATGATGGACAATTACATAGTACCCATAACAGCCATGCACTGTCCAAAGC 82 ATGCCCTTGTGCACAGGACAGCAA (SEO ID NO: 23 O) His MT1 GTCGGCATCTGAGACCAGTGAGAAACGCCCCTTCATGTGTGCTTACCCAGGCTGCAAT 149 AAGAGATATTTTAAGCTGTCCCACTTAGAGATGCACAGGAGGAAGCACACTGGTGAGA AACCATACCAGTGTGACTTCAAGGACTGTGAAC (SEQ ID NO. 231) Hs ZWINT CCCAGAGGAAACGGACACAACTCCGGGAGCCTTTGAGCAGCTCCAGGCCAAGAACA 124 AATGGCCATGGAGAAACGCAGAGCACTCCACAACCAGTGGCAGCTACAACAGGAGAAG CATCTGCA (SEO ID NO. 232)

(2) indicates text missing or illegible when filed

0093. For subsequent qPCR analysis, 121 genes that lis, Wilcoxon-Mann-Whitney or Student's t-test) higher in encode molecules in the following categories were selected LSCs than in HSCs. The columns in Table 2 indicates, in the from among the 217 genes identified in microarray experi order from the left column, , Gene ID (Column A), ments: HUGO Gene Symbol (Column B) localization (Column C), 1) Those located on the cell membrane or in extracellular molecular function (Column D), biological process (Column Spaces, E), P values from each statistical test (Columns F-H), ratio of 2) , growth factors, transmembranous receptors, median values of the mRNA levels (Column I), and the num protein kinases, phosphatases, transcriptional regulation ber of LSC samples showing a higher expression level than molecules, and/or signaling molecules, and the mRNA levels for the HSC samples (Column J). 3) those involved in immune regulation, cell cycle, apoptosis, 0.095 The present inventors previously reported that LSCs and/or cell adhesion. derived from bone marrow (BM) of AML patient origin and 0094. The list includes 57 genes, the mRNA levels of LSCs derived from BM of a mouse receiving transplantation which were significantly (P<0.05; according to Kruskal-Wal of AML patient BM have similar transcription profiles (Na US 2012/0070450 A1 Mar. 22, 2012 27 ture Biotechnology, 2007, ibid). Based on this finding, the antibodies proven to be effective and 32 AML patient present inventors performed a comprehensive transcriptome samples. Through the flow cytometric analysis, the following analysis to compare LSCs and normal hematopoietic stem aspects were examined in each candidate molecule: 1) local cells (HSCs), using two array platforms: Human Genome ization (on cell Surfaces or in cells), 2) frequency of positive U133 plus 2.0 GeneChips (BMderived from 16 AML patients cells, and 3) expression intensity. Out of the 57 candidate and BM derived from 5 AML transplantation recipient mice molecules thus assessed, FCGR2A(CD32), ITGB2(CD18), were compared with BM derived from 2 healthy donors and CD93, CD33, CD3D and TNF(TNFa) were found to have the cord blood (CB) derived from 5 healthy donors) and Human most promising expression level/pattern for LSC-specific Gene 1.0ST GeneChips (BM derived from 1 AML patient and markers/targets. In particular, the expression of FCGR2A BM derived from 5 AML transplantation recipient mice were compared with CB from 1 healthy donor and BM from 4). (CD32) exhibited a strong correlation with LSCs in a signifi Since a previous study had revealed that AML stem cells are cant ratio of the AML patients tested, and this was selected for present exclusively in the CD34+CD38-fraction, >1.2x10' further functional analysis. In 9 of the 32 AML patients tested, CD34+CD38-cells were recovered with a purity of >98% the great majority (>80%) of AML stem cells expressed this (FIG. 1). Using the same method, CD34+CD38-HSCs were antigen (FIG. 3). To confirm that the expression of CD32 also purified from normal BM and CB samples (FIG. 1). By correlates exclusively with the function, in vivo NOD/SCID/ intravenously injecting the aforementioned purified HSCs IL2rg KO transplantation assay was performed using purified and LSCs to neonatal NOD/SCID/IL2rg KO mice, the onset LSCs derived from three patients with AML. When purified of AML by LSCs and the lack of reconstitution of normal CD34+CD38-CD32+ and CD34+CD38-CD32- cells were immunity were confirmed, and long-time transplantation and transplanted to sub-lethally irradiated recipients, AML devel multi-lineage (T/B/bone marrow) differentiation of HSCs oped exclusively from the CD32+ fraction (FIG. 4). Because were confirmed (FIG. 1). Not the CD34+CD38+ cells or any LSC-targeting treatment is thought to be best used along CD34-cells derived from the AML transplantation recipient with a commonly used chemotherapeutic agent that is effec mice, but the CD34+CD38- bone marrow cells caused leu tive in removing non-LSC AML cells, it is important to con kemia in secondary recipients. These data Suggest that the firm that the target molecule is continuously expressed even transplanted CD34+CD38- cells did not come from the after chemotherapy. Accordingly, the present inventors exam HSCs, but retained the nature of the LSCs. To analyze the ined whether the expression of CD32 was maintained after expression data set obtained, genes that exhibit a significantly chemotherapy, and confirmed the expression of CD32 in BM, higher (p value <0.01, percentage of false positively <0.05) spleens and peripheral blood (PB) of AML transplantation array signal in LSCs than in HSCs on both the two microarray platforms were extracted using RankProd (Bioinformatics recipient mice after AraC treatment (FIG. 6). Also, CD32 22, 2825, 2006) mounted on the Bioconductor package. A expressing cells were found by immunofluorescent labeling total of 217 gene candidates met the criteria (FIG. 5, Table 1); in both the membrane region and central region of bone further, IL2R was added to make a total of 218 gene candi marrow (FIG. 4). This finding, in view of the previous report dates. by the present inventors that chemotherapy-resistant LSCs 0096. Next, to demonstrate the expression levels of candi are present in BMosteoblast niches, further supports CD32 as dates for separating LSCs and HSCs, quantitative PCR a candidate for LSC target therapy (Ishikawa F. et al. Nature (qPCR) was performed for each candidate gene using LSCs Biotechnol 25:1315-1321, 2007 and PCT/JP2008/068892). derived from BM of 5 AML patients and HSCs derived from (0099 Next, the expression of CD32 in normal human BM of 4 (Table 3). Out of the 217 genes identified, 121 genes HSCs was assessed. In the primary human CB CD34+CD38 that encode molecules in the following categories were population, the frequency of CD32+ cells was 9.8%+/-SD selected as candidates best Suiting for the development of (FIG.4A). When the expression of CD133 in this fraction was pharmaceuticals, and Subjected to Subsequent analysis. The analyzed, CD32+ cells were detected exclusively in the three categories are as follows: CD34+CD38-CD133- fraction (FIG. 4a). It was found by 1) those located on the cell membrane or in extracellular heterologous transplantation assay that not the CD34+ Spaces, CD38-CD32+ fraction but the CD34+CD38-CD133+ 2) cytokines, growth factors, transmembranous receptors, CD32- fraction contains HSCs (FIG. 4B). Furthermore, it protein kinases, phosphatases, transcriptional regulation was suggested by in vitro colony-forming cell (CFC) assay molecules, and/or signaling molecules, and that not CD34+CD38-CD32+ cells but CD34+CD38 3) those involved in immune regulation, cell cycle, apoptosis, CD32-cells have the capability of producing bone marrow and/or cell adhesion. series and erythrocyte-series hematopoietic colonies. The 0097. As shown in Table 2, the mRNA contents concern lack of the capability of in vivo long-term hematopoiesis ing 57 genes out of the 121 genes were statistically higher in reconstitution in CD32+ normal HSCs suggests the possibil LSCs than in HSCs. Of the 57 genes, 35 genes were identified ity that therapeutic agents targeting CD32 expression cells as excellent LSC markers. The reason was that 1) the median may help avoid important adverse reactions related to the expression levels of these genes were 5 times or higher in normal hematopoietic and immune systems without affecting LSCs, and that 2) their mRNA contents were higher in all HSCS LSC samples tested than in each HSC population tested (FIG. 0100. The present inventors first confirmed by neonatal 2). NOD/SCID/IL2rg KO mouse transplantation assay that in 0098. To confirm the expression of these LSC-specific AML patient samples lacking the expression of CD32 by candidate molecules at the protein level, the quality of mono LSCs, CD34+CD38-cells possess the LSC function (FIG. clonal antibodies and polyclonal antibodies that can be uti 1), and then examined the expression of ITGB2(CD18), lized for the 35 candidate molecules, respectively, was veri CD93, (as well as CD25, CD132, OX41, and CD97), CD33, fied, and flow cytometric analysis was performed using CD3D and TNFC. by flow cytometry. Combination of the US 2012/0070450 A1 Mar. 22, 2012 28 antigens CD32, ITGB2, CD93, 97 and 33 enabled good sepa 0106 WT1 has been shown to be expressed in a wide ration of LSCs from normal HSCs in 31 patients out of 47 variety of tumors, including leukemia. However, whether this patients. molecule is expressed at the level of stem cells, which exhibit 0101 The list of LSC-specific genes identified using the recurrences and anticancer agent resistance, has been two sets of microarrays and quantitative PCR (Table 2) includes genes that are expressed preferentially in bone mar unknown. The present inventors found that this molecule is row progeny, but their expression is limited in HSCs. For expressed in leukemic stem cells that are present in niches and example, FCGR2A, HCK and NCF4 are highly expressed in are in the stationary phase of cell cycle, and have shown that mature bone marrow cells and mediate the phagocytosis by the molecule is of significance as a target molecule for killing immunoconjugates and Subsequent Superoxide production leukemic stem cells, which have been unable to be killed by (Prot Natl Acad Sci USA, 97, 1725; 2000; J Exp Med 191, conventional chemotherapy and radiotherapy. 669, 2000; Nat Cell Biol, 3, 679, 2001; J Biol Chem 279, 0107 Also, peripheral blood was collected from 47 1415, 2004). Meanwhile, CD3D, which is a constituent of the patients with AML in various stages, samples containing CD3 conjugate, transmits in mature T lymphocytes a hematopoietic cells were prepared, and FCGR2A(CD32a), receptor signal via the ITAM motifthereof. Therefore, at least a particular ratio of AMLS can develop via abnormal regula FCGR2B(CD32b), IL2RA(CD25), ITGB2(CD18) and tion of differentiation in the stem cell stage. CD93 positively rates in leukemic stem cells contained in the 0102 Another feature of the list is the involvement of samples were examined. The results are shown in Table 4. genes expressed remarkably in cancer cells and leukemia cells. For example, CD33 is a well recognized immunological TABLE 4 marker of AML cells, and serves as a target for antibody Any pharmaceuticals such as gemtuzumab ozogamicin (Leuke in marker CD32-a CD32-b CD25 CD18 CD93 mia 19: 176, 2005). Furthermore, CD97 has been reported to AML MO 2 2 2 O O O O be accumulated in colorectal cancers that infiltrate lymphatic % marker positive 1OO.O 1OO.O O.O O.O O.O O.O vessels (Am Pathol 161, 1657, 2002). Overexpression of AML M1 7 4 O 2 2 3 O these molecules in LSCs suggests that a therapeutic method % marker positive 57.1 O.O 28.6 28.6 42.9 O.O that targets these molecules may be effective not only on AML M2 14 9 5 4 4 5 1 % marker positive 64.3 35.7 28.6 28.6 35.7 7.1 LSCs, but also on mature AML cells. It should be noted that AML M4 4 4 3 1 1 2 1 gene products of BIK, HOMER3, WT1 (Genes Chromo % marker positive 1OO.O 75.0 25.0 25.O SO.O 25.0 somes Cancer 47, 8-20, 2008) and CLEC12A (encoding Other AML 3 1 1 O O O O % marker positive 33.3 33.3 O.O O.O O.O O.O CLL-1)(Blood 110,2659-2666, 2007) have been proposed as MDSAML 17 11 3 6 9 O 1 marker molecules for LSC/AML blasts, and this demon % marker positive 64.7 17.6 35.3 S2.9 O.O 5.9 strates that the findings of the present invention agree with All cases 47 31 14 13 16 10 3 available reports. % marker positive 66.O 29.8 27.7 34.O 213 6.4 0103). By analyzing the expression levels/patterns, the candidate genes were classified into the following sets: 1) a set of genes that encode molecules expressed in a signifi 0108. From Table 4, it is seen that by combining 4kinds of cant ratio of LSCs at the RNA and protein levels, but markers FCGR2A(CD32a), IL2RA(CD25), ITGB2(CD18) expressed in only a small ratio of HSCs (or not expressed), and CD93, leukemic stem cells can be distinguished at a high and rate, and by combining pharmaceuticals that target these 4 2) a set of genes expressed at the protein level in LSCs and kinds of genes, over 60% of leukemia cells can be extermi HSCs, but whose expression intensity as determined by flow nated. cytometry allows separation of LSCs from HSCs. 0104. The gene set 1 includes candidates that specifically INDUSTRIAL APPLICABILITY target LSCs and do not affect HSCs, for example, promising candidates for the development of therapeutic agents such as 0109. By using a leukemic stem cell marker found in the antibody pharmaceuticals based on the lack of the aforemen present invention as a molecular target, a therapeutic agent tioned candidates in normal HSCs. The genes included in the that acts specifically on. LSCs that are the source of onset or gene set 2 (the most promising candidate is CD33) encode recurrence of AML can be provided. It is possible to specifi biomarkers having high applicability to ex vivo purging of cally remove LSCs from bone marrow cells of a patient or a LSCs for separating LSCs from HSCs and the like against the donor using a cell sorter Such as FACS, with a leukemic stem background of autologous transplantation of hematopoietic cell marker found in the present invention as an index. This stem cells. will increase effectiveness of purging for autologous or allo 0105. As shown in FIG. 7, it was found by identifying the geneic bone marrow transplantation, and enable to signifi location and the phase in cell cycle by imaging using an antibody against each marker (FCGR2A, AK5, DOK2, cantly prevent recurrences or the initial onset of acute LRG1, BIK, IL2RA, WT1 and SUCNR1) and a stationary myeloid leukemia. Furthermore, the presence or absence of cell-specific marker, that these molecules are abundantly LSCs in a collected biological sample or in a living organism present in the endosteum (niches), where stem cells exhibit can be determined with a leukemic stem cell marker found in ing anticancer agent resistance are present, and are expressed the present invention as an index, whereby recurrences or the in leukemic stem cells while in the stationary phase of cell initial onset of acute myeloid leukemia can also be predicted. cycle. Therefore, targeting these individual marker molecules 0110. This application is based on a patent application No. is thought to be largely contributory to overcoming recur 2009-072400 filed in Japan (filing date: Mar. 24, 2009), the rences of leukemia. contents of which are incorporated in full herein. US 2012/0070450 A1 Mar. 22, 2012 29

SEQUENCE LISTING

<16O is NUMBER OF SEO ID NOS: 232

<210s, SEQ ID NO 1 &211s LENGTH: 24 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Synthetic Probe <4 OOs, SEQUENCE: 1 t cctggcctg. C catcacggit ta 24

<210s, SEQ ID NO 2 &211s LENGTH: 24 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Synthetic Primer <4 OOs, SEQUENCE: 2 gtgctttctg gagggit ctac tatg 24

<210s, SEQ ID NO 3 &211s LENGTH: 21 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Synthetic Primer <4 OOs, SEQUENCE: 3 ggtgggtctt caatgcggat C 21

<210s, SEQ ID NO 4 &211s LENGTH: 26 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Synthetic Probe <4 OOs, SEQUENCE: 4 actgatgagt cccactgtgc tigagca 26

<210s, SEQ ID NO 5 &211s LENGTH: 23 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Synthetic Primer <4 OOs, SEQUENCE: 5 gtagagtgga aaaggagc at td 23

<210s, SEQ ID NO 6 &211s LENGTH: 22 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Synthetic Primer <4 OOs, SEQUENCE: 6 tacaccittgg atgttggaca gg 22 US 2012/0070450 A1 Mar. 22, 2012 30

- Continued

<210s, SEQ ID NO 7 &211s LENGTH: 26 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Synthetic Probe <4 OO > SEQUENCE: 7 cct catcct c atcgcggit cq goatca 26

<210s, SEQ ID NO 8 &211s LENGTH: 25 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Synthetic Primer <4 OOs, SEQUENCE: 8 gctgct coat tdgittaaata citt co 25

<210s, SEQ ID NO 9 &211s LENGTH: 22 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Synthetic Primer < 4 OO > SEQUENCE: 9 gttgtcaact gcc atgctga tig 22

<210s, SEQ ID NO 10 &211s LENGTH: 26 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Synthetic Probe <4 OOs, SEQUENCE: 10 agaacgcaga gcacccCtgg ctacat 26

<210s, SEQ ID NO 11 &211s LENGTH: 24 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Synthetic Primer <4 OOs, SEQUENCE: 11 agtactttgt C9gttacct a ggag 24

<210s, SEQ ID NO 12 &211s LENGTH: 25 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Synthetic Primer <4 OOs, SEQUENCE: 12 gtaatagttgaatatgc.cag caacg 25

<210s, SEQ ID NO 13 &211s LENGTH: 28 &212s. TYPE: DNA US 2012/0070450 A1 Mar. 22, 2012 31

- Continued <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Synthetic Probe <4 OOs, SEQUENCE: 13 t caggctgtc. cagaat ctitc caaccaag 28

<210s, SEQ ID NO 14 &211s LENGTH: 23 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Synthetic Primer <4 OOs, SEQUENCE: 14 gct cagtgtg gagtat ct ca aag 23

<210s, SEQ ID NO 15 &211s LENGTH: 21 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Synthetic Primer <4 OOs, SEQUENCE: 15 citt cagtgtg tocctgtttg c 21

<210s, SEQ ID NO 16 &211s LENGTH: 22 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Synthetic Probe <4 OOs, SEQUENCE: 16 cgc.ctggc cc agctict Coga gg 22

<210s, SEQ ID NO 17 &211s LENGTH: 2O &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Synthetic Primer <4 OOs, SEQUENCE: 17 agatggacgt gag cct cagg

<210s, SEQ ID NO 18 &211s LENGTH: 23 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Synthetic Primer <4 OOs, SEQUENCE: 18 t cagtctggit cqtagatgaa agc 23

<210s, SEQ ID NO 19 &211s LENGTH: 26 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Synthetic Probe US 2012/0070450 A1 Mar. 22, 2012 32

- Continued <4 OOs, SEQUENCE: 19 agcgaatgag gcacagcaac CactCc 26

<210s, SEQ ID NO 2 O &211s LENGTH: 23 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Synthetic Primer <4 OOs, SEQUENCE: 2O ccacagcaat ttctgggitta cag 23

<210s, SEQ ID NO 21 &211s LENGTH: 22 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Synthetic Primer <4 OOs, SEQUENCE: 21 gacaag.cggit toc tact citt cc 22

<210s, SEQ ID NO 22 &211s LENGTH: 26 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Synthetic Probe <4 OOs, SEQUENCE: 22 aacccagoag togtotttgt caccc.g 26

<210s, SEQ ID NO 23 &211s LENGTH: 25 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Synthetic Primer <4 OOs, SEQUENCE: 23 t caaggagta tttctacacic agtgg 25

<210s, SEQ ID NO 24 &211s LENGTH: 22 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Synthetic Primer <4 OOs, SEQUENCE: 24 tcc.cgaac cc atttcttct c td 22

<210s, SEQ ID NO 25 &211s LENGTH: 26 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Synthetic Probe <4 OOs, SEQUENCE: 25 taccacaggg toagcct c co cqaaac 26 US 2012/0070450 A1 Mar. 22, 2012 33

- Continued

<210s, SEQ ID NO 26 &211s LENGTH: 2O &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Synthetic Primer <4 OOs, SEQUENCE: 26 Cagcagtggg Caggaatgac

<210s, SEQ ID NO 27 &211s LENGTH: 23 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Synthetic Primer <4 OOs, SEQUENCE: 27 t cct catcca tot coacagt agg 23

<210s, SEQ ID NO 28 &211s LENGTH: 26 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Synthetic Probe < 4 OO > SEQUENCE: 28 tgttcc cacc gttcc.cticta cc catg 26

<210s, SEQ ID NO 29 &211s LENGTH: 22 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Synthetic Primer <4 OOs, SEQUENCE: 29 tgg tactggc taccct tctic tic 22

<210s, SEQ ID NO 3 O &211s LENGTH: 24 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Synthetic Primer <4 OOs, SEQUENCE: 30 tccagt cittg taatgtctga cagc 24

<210s, SEQ ID NO 31 &211s LENGTH: 26 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Synthetic Probe <4 OOs, SEQUENCE: 31 agggccacct cactitt cagc agtctg 26

<210s, SEQ ID NO 32 &211s LENGTH: 21 &212s. TYPE: DNA US 2012/0070450 A1 Mar. 22, 2012 34

- Continued <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Synthetic Primer <4 OOs, SEQUENCE: 32 aatgcggcag acagtt actic C 21

<210s, SEQ ID NO 33 &211s LENGTH: 21 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Synthetic Primer <4 OOs, SEQUENCE: 33 gtggctggtg actictagtgt C 21

<210s, SEQ ID NO 34 &211s LENGTH: 25 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Synthetic Probe <4 OOs, SEQUENCE: 34 cgcct tcc to tacctgctgc actgc 25

<210s, SEQ ID NO 35 &211s LENGTH: 25 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Synthetic Primer <4 OOs, SEQUENCE: 35 citatgtgttt accatcctica actgc 25

<210s, SEQ ID NO 36 &211s LENGTH: 21 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Synthetic Primer <4 OOs, SEQUENCE: 36 gcc.cact tcc gg tatt ctitc c 21

<210s, SEQ ID NO 37 &211s LENGTH: 30 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Synthetic Probe <4 OO > SEQUENCE: 37 cct citccacc acactgcaaa caatagocac 3 O

<210s, SEQ ID NO 38 &211s LENGTH: 26 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Synthetic Primer US 2012/0070450 A1 Mar. 22, 2012 35

- Continued <4 OOs, SEQUENCE: 38 acatgaatat citccaacaag at cagg 26

<210s, SEQ ID NO 39 &211s LENGTH: 23 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Synthetic Primer <4 OOs, SEQUENCE: 39 gctgtc. citta tdccaaatcc atc 23

<210s, SEQ ID NO 4 O &211s LENGTH: 25 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Synthetic Probe <4 OOs, SEQUENCE: 4 O Ccagcgcgat gtcaactgct cqgtt 25

<210s, SEQ ID NO 41 &211s LENGTH: 19 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Synthetic Primer <4 OOs, SEQUENCE: 41 tctt coaggt gggctic cag 19

<210s, SEQ ID NO 42 &211s LENGTH: 21 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Synthetic Primer <4 OOs, SEQUENCE: 42 gccagaattig C caagg to at C 21

<210s, SEQ ID NO 43 &211s LENGTH: 26 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Synthetic Probe <4 OOs, SEQUENCE: 43 tgccaa.cagg gtcacagcca ggtaca 26

<210s, SEQ ID NO 44 &211s LENGTH: 26 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Synthetic Primer <4 OOs, SEQUENCE: 44 tgat cott at t cagtag cact citctg 26 US 2012/0070450 A1 Mar. 22, 2012 36

- Continued

<210s, SEQ ID NO 45 &211s LENGTH: 21 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Synthetic Primer <4 OOs, SEQUENCE: 45 agcgtgatga caactic cagt g 21

<210s, SEQ ID NO 46 &211s LENGTH: 24 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Synthetic Probe <4 OOs, SEQUENCE: 46

Cctict c caga gacgcagcga C9gc 24

<210s, SEQ ID NO 47 &211s LENGTH: 2O &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Synthetic Primer < 4 OO > SEQUENCE: 47 agctgtacga Ctggcc ctac

<210s, SEQ ID NO 48 &211s LENGTH: 21 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Synthetic Primer <4 OOs, SEQUENCE: 48 tgc.cgggttt Caact caaa g 21

<210s, SEQ ID NO 49 &211s LENGTH: 26 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Synthetic Probe <4 OOs, SEQUENCE: 49 agcaccagga accaggagaci ttacga 26

<210s, SEQ ID NO 50 &211s LENGTH: 24 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Synthetic Primer <4 OOs, SEQUENCE: 50 gagaaatcag atggtgttta cacg 24

<210s, SEQ ID NO 51 &211s LENGTH: 22 &212s. TYPE: DNA US 2012/0070450 A1 Mar. 22, 2012 37

- Continued <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Synthetic Primer <4 OOs, SEQUENCE: 51 c tactgtggt gigtttct cat gc 22

<210s, SEQ ID NO 52 &211s LENGTH: 26 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Synthetic Probe <4 OOs, SEQUENCE: 52 tgtc.ccagaa acctgtggct gct tca 26

<210s, SEQ ID NO 53 &211s LENGTH: 24 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Synthetic Primer <4 OOs, SEQUENCE: 53 gatgactatg gagaccCaaa titc 24

<210s, SEQ ID NO 54 &211s LENGTH: 22 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Synthetic Primer <4 OOs, SEQUENCE: 54 caagttt cag cacago ctitt gg 22

<210s, SEQ ID NO 55 &211s LENGTH: 26 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Synthetic Probe <4 OO > SEQUENCE: 55 cc.cagtagtt to acct ctdt togc.ccc 26

<210s, SEQ ID NO 56 &211s LENGTH: 23 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Synthetic Primer <4 OOs, SEQUENCE: 56 titt cittaaat gg.ccca catc agg 23

<210s, SEQ ID NO 57 &211s LENGTH: 23 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Synthetic Primer US 2012/0070450 A1 Mar. 22, 2012 38

- Continued <4 OO > SEQUENCE: 57 gaaatcCagt taagtggctg. tcc 23

<210s, SEQ ID NO 58 &211s LENGTH: 26 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Synthetic Probe <4 OOs, SEQUENCE: 58 agccaaccac catgaaag.ca tot cac 26

<210s, SEQ ID NO 59 &211s LENGTH: 2O &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Synthetic Primer <4 OO > SEQUENCE: 59 accaccitcCa CCatcc catc.

<210s, SEQ ID NO 60 &211s LENGTH: 26 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Synthetic Primer <4 OOs, SEQUENCE: 60 acac catctt gattgtctitc attgac 26

<210s, SEQ ID NO 61 &211s LENGTH: 26 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Synthetic Probe <4 OOs, SEQUENCE: 61 tggcct tact c ct cocacagaatgcg 26

<210s, SEQ ID NO 62 &211s LENGTH: 26 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Synthetic Primer <4 OOs, SEQUENCE: 62 cataccataa caatgacgac aacttic 26

<210s, SEQ ID NO 63 &211s LENGTH: 24 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Synthetic Primer <4 OOs, SEQUENCE: 63

Catgggacag gtagtaac at ttgg 24 US 2012/0070450 A1 Mar. 22, 2012 39

- Continued

SEQ ID NO 64 LENGTH: 26 TYPE: DNA ORGANISM: Artificial Sequence FEATURE: OTHER INFORMATION: Synthetic Probe

SEQUENCE: 64 agcc.ca.gcac agacticatgg tag cag 26

SEO ID NO 65 LENGTH: 24 TYPE: DNA ORGANISM: Artificial Sequence FEATURE: OTHER INFORMATION: Synthetic Primer

SEQUENCE: 65 ctittgggtga gttgaactitc titcc 24

SEQ ID NO 66 LENGTH: 22 TYPE: DNA ORGANISM: Artificial Sequence FEATURE: OTHER INFORMATION: Synthetic Primer SEQUENCE: 66 cc.ca.gctaac togcaa.cataa cq 22

SEO ID NO 67 LENGTH: 26 TYPE: DNA ORGANISM: Artificial Sequence FEATURE: OTHER INFORMATION: Synthetic Probe SEQUENCE: 67 accct cqctt cagccacagt titcctic 26

SEQ ID NO 68 LENGTH: 21 TYPE: DNA ORGANISM: Artificial Sequence FEATURE: OTHER INFORMATION: Synthetic Primer SEQUENCE: 68 c cct tcctac toccagacac c 21

SEO ID NO 69 LENGTH: 24 TYPE: DNA ORGANISM: Artificial Sequence FEATURE: OTHER INFORMATION: Synthetic Primer SEQUENCE: 69 agagattitt c cagtccaa.cc tacc 24

SEO ID NO 7 O LENGTH: 24 TYPE: DNA US 2012/0070450 A1 Mar. 22, 2012 40

- Continued <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Synthetic Probe <4 OO > SEQUENCE: 7 O cctgcttittg citcc.ca.gtgg ctgc 24

<210s, SEQ ID NO 71 &211s LENGTH: 22 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Synthetic Primer <4 OOs, SEQUENCE: 71 gatc catctg gigt cacatcc tic 22

<210s, SEQ ID NO 72 &211s LENGTH: 21 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Synthetic Primer <4 OOs, SEQUENCE: 72

Cagggitaaaa gCagggagt g 21

<210s, SEQ ID NO 73 &211s LENGTH: 28 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Synthetic Probe <4 OO > SEQUENCE: 73 tgttcc ctitg tagctg.cgtc ctittacca 28

<210s, SEQ ID NO 74 &211s LENGTH: 26 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Synthetic Primer <4 OOs, SEQUENCE: 74 gccatgaatt tacct citat gaaaac 26

<210s, SEQ ID NO 75 &211s LENGTH: 24 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Synthetic Primer <4 OO > SEQUENCE: 75 gctgacattt gatgcc actic ttag 24

<210s, SEQ ID NO 76 &211s LENGTH: 26 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Synthetic Probe US 2012/0070450 A1 Mar. 22, 2012 41

- Continued <4 OO > SEQUENCE: 76 Cctt.cgtgag cacagcatag ggacct 26

<210s, SEQ ID NO 77 &211s LENGTH: 23 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Synthetic Primer <4 OO > SEQUENCE: 77 cagttcagca toagttccaa gac 23

<210s, SEQ ID NO 78 &211s LENGTH: 19 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Synthetic Primer <4 OO > SEQUENCE: 78 ttgtacgt.ct gcggcatgg 19

<210s, SEQ ID NO 79 &211s LENGTH: 23 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Synthetic Probe <4 OO > SEQUENCE: 79 tcgc.ccacgg agc cct caga caa 23

<210s, SEQ ID NO 8O &211s LENGTH: 2O &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Synthetic Primer <4 OOs, SEQUENCE: 80 aaactgttcc gcagdcagag 2O

<210s, SEQ ID NO 81 &211s LENGTH: 2O &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Synthetic Primer <4 OOs, SEQUENCE: 81 aact cqgcct c cc actgtac 2O

<210s, SEQ ID NO 82 &211s LENGTH: 26 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Synthetic Probe <4 OOs, SEQUENCE: 82 t cagc.cagtic cct tag acac accact 26 US 2012/0070450 A1 Mar. 22, 2012 42

- Continued

<210s, SEQ ID NO 83 &211s LENGTH: 26 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Synthetic Primer <4 OOs, SEQUENCE: 83 cctagaggat cittgttactgaat acc 26

<210s, SEQ ID NO 84 &211s LENGTH: 22 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Synthetic Primer <4 OOs, SEQUENCE: 84 agagtcgttc actgtagt ct gg 22

<210s, SEQ ID NO 85 &211s LENGTH: 22 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Synthetic Probe < 4 OO > SEQUENCE: 85 ttgc.ccgcct c ccagaccac ca 22

<210s, SEQ ID NO 86 &211s LENGTH: 21 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Synthetic Primer <4 OOs, SEQUENCE: 86 cc.cgcatc cc ticacatgaaa g 21

<210s, SEQ ID NO 87 &211s LENGTH: 21 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Synthetic Primer <4 OO > SEQUENCE: 87 agt caccaga cacticcitcca g 21

<210s, SEQ ID NO 88 &211s LENGTH: 25 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Synthetic Probe <4 OOs, SEQUENCE: 88 tctgat coac Ctgagcgacc ticcgg 25

<210s, SEQ ID NO 89 &211s LENGTH: 21 &212s. TYPE: DNA US 2012/0070450 A1 Mar. 22, 2012 43

- Continued <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Synthetic Primer <4 OOs, SEQUENCE: 89 t cct gctggit Catctggaag g 21

<210s, SEQ ID NO 90 &211s LENGTH: 22 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Synthetic Primer <4 OOs, SEQUENCE: 90

Cagcaaactt ggggitt catg ac 22

<210s, SEQ ID NO 91 &211s LENGTH: 29 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Synthetic Probe <4 OOs, SEQUENCE: 91 titcgitatic catctggcagct togacggtca 29

<210s, SEQ ID NO 92 &211s LENGTH: 18 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Synthetic Primer <4 OOs, SEQUENCE: 92 gcgggaggct gtc.ttt CC 18

<210s, SEQ ID NO 93 &211s LENGTH: 18 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Synthetic Primer <4 OOs, SEQUENCE: 93

Caggttgagg C9gttggg 18

<210s, SEQ ID NO 94 &211s LENGTH: 26 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Synthetic Probe <4 OOs, SEQUENCE: 94 tgc.ca.gcatc togct ct cact gcaacc 26

<210s, SEQ ID NO 95 &211s LENGTH: 2O &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Synthetic Primer US 2012/0070450 A1 Mar. 22, 2012 44

- Continued <4 OO > SEQUENCE: 95 tgaa.gc.ccaa gagccalaagg

<210s, SEQ ID NO 96 &211s LENGTH: 21 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Synthetic Primer <4 OOs, SEQUENCE: 96 t cct gcttgt Ctggtaagtg C 21

<210s, SEQ ID NO 97 &211s LENGTH: 25 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Synthetic Probe <4 OO > SEQUENCE: 97 agaccttgcc acct gacctic ctdag 25

<210s, SEQ ID NO 98 &211s LENGTH: 2O &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Synthetic Primer <4 OOs, SEQUENCE: 98 CCttgacctt ggggagaacc

<210s, SEQ ID NO 99 &211s LENGTH: 25 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Synthetic Primer <4 OOs, SEQUENCE: 99 ttctagatgt agcc.gttcta attgc 25

<210s, SEQ ID NO 100 &211s LENGTH: 26 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Synthetic Probe <4 OOs, SEQUENCE: 1.OO citat cocatc titgaggcgg Ctctgc 26

<210s, SEQ ID NO 101 &211s LENGTH: 24 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Synthetic Primer <4 OOs, SEQUENCE: 101 atgtct caag gct catctgt tttg 24 US 2012/0070450 A1 Mar. 22, 2012 45

- Continued

<210s, SEQ ID NO 102 &211s LENGTH: 23 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Synthetic Primer <4 OOs, SEQUENCE: 102 tgacaggc cc agcatagitaa atc 23

<210s, SEQ ID NO 103 &211s LENGTH: 27 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Synthetic Probe <4 OOs, SEQUENCE: 103 ttggct cotg gaccatatt c tictdggit 27

<210s, SEQ ID NO 104 &211s LENGTH: 27 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Synthetic Primer < 4 OO > SEQUENCE: 104 atatt catgt tttacaagga gaaac cc 27

<210s, SEQ ID NO 105 &211s LENGTH: 26 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Synthetic Primer <4 OOs, SEQUENCE: 105 agatttgctt totgtctato acaatg 26

<210s, SEQ ID NO 106 &211s LENGTH: 24 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Synthetic Probe <4 OOs, SEQUENCE: 106 cgttgaccgc atggcagctic cag 24

<210s, SEQ ID NO 107 &211s LENGTH: 18 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Synthetic Primer <4 OOs, SEQUENCE: 107 agagcgtgtc. cccacagg 18

<210s, SEQ ID NO 108 &211s LENGTH: 23 &212s. TYPE: DNA US 2012/0070450 A1 Mar. 22, 2012 46

- Continued <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Synthetic Primer <4 OOs, SEQUENCE: 108 cgactgagga ggalagat cac atc 23

<210s, SEQ ID NO 109 &211s LENGTH: 26 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Synthetic Probe <4 OOs, SEQUENCE: 109 actggt cagc atgtgcatct gcc ctg 26

<210s, SEQ ID NO 110 &211s LENGTH: 21 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Synthetic Primer <4 OOs, SEQUENCE: 110 ggggagcact act cog agaia g 21

<210s, SEQ ID NO 111 &211s LENGTH: 22 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Synthetic Primer <4 OOs, SEQUENCE: 111 cacgitatic cq atgtcagg to tc 22

<210s, SEQ ID NO 112 &211s LENGTH: 30 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Synthetic Probe <4 OOs, SEQUENCE: 112 acgcttacca togtaaaggc acgtccaatt 3 O

<210s, SEQ ID NO 113 &211s LENGTH: 26 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Synthetic Primer <4 OOs, SEQUENCE: 113 agaggittata atctgaatcc caaagg 26

<210s, SEQ ID NO 114 &211s LENGTH: 26 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Synthetic Primer US 2012/0070450 A1 Mar. 22, 2012 47

- Continued <4 OOs, SEQUENCE: 114 taaaggagtic attatagaag cagtgg 26

<210s, SEQ ID NO 115 &211s LENGTH: 26 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Synthetic Probe <4 OOs, SEQUENCE: 115 tccacccaag gct ctdcgac titc cat 26

<210s, SEQ ID NO 116 &211s LENGTH: 21 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Synthetic Primer <4 OOs, SEQUENCE: 116 gact acagca acgaggagca C 21

<210s, SEQ ID NO 117 &211s LENGTH: 25 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Synthetic Primer <4 OOs, SEQUENCE: 117 ggtc.gctctg cataaaat at tcc to 25

<210s, SEQ ID NO 118 &211s LENGTH: 24 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Synthetic Probe <4 OOs, SEQUENCE: 118 tcgtgttgag accticcc.gcg cagt 24

<210s, SEQ ID NO 119 &211s LENGTH: 24 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Synthetic Primer <4 OOs, SEQUENCE: 119 acatgt attcaactgcacca agac 24

<210s, SEQ ID NO 120 &211s LENGTH: 22 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Synthetic Primer <4 OOs, SEQUENCE: 120 tgggcaatcc at aaccaaaa cc 22 US 2012/0070450 A1 Mar. 22, 2012 48

- Continued

SEQ ID NO 121 LENGTH: 27 TYPE: DNA ORGANISM: Artificial Sequence FEATURE: OTHER INFORMATION: Synthetic Probe

SEQUENCE: 121 ttgcc.gtagg toccactgtt gttcttgc 27

SEQ ID NO 122 LENGTH: 22 TYPE: DNA ORGANISM: Artificial Sequence FEATURE: OTHER INFORMATION: Synthetic Primer

SEQUENCE: 122 gatcagactic agcctic ct ca ga 22

SEQ ID NO 123 LENGTH: 18 TYPE: DNA ORGANISM: Artificial Sequence FEATURE: OTHER INFORMATION: Synthetic Primer SEQUENCE: 123 gctgctg.ccc ticccagat 18

SEQ ID NO 124 LENGTH: 26 TYPE: DNA ORGANISM: Artificial Sequence FEATURE: OTHER INFORMATION: Synthetic Probe SEQUENCE: 124 agaccc ct co toggcc.gctac tottitt 26

SEQ ID NO 125 LENGTH 19 TYPE: DNA ORGANISM: Artificial Sequence FEATURE: OTHER INFORMATION: Synthetic Primer SEQUENCE: 125 tggcc.cagag tdcatgtc. 19

SEQ ID NO 126 LENGTH: 2O TYPE: DNA ORGANISM: Artificial Sequence FEATURE: OTHER INFORMATION: Synthetic Primer SEQUENCE: 126 ggct Cagg taggct cagacic

SEO ID NO 127 LENGTH: 26 TYPE: DNA US 2012/0070450 A1 Mar. 22, 2012 49

- Continued <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Synthetic Probe <4 OOs, SEQUENCE: 127

Ccagcacctic gcatcagcca gagttg 26

<210s, SEQ ID NO 128 &211s LENGTH: 22 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Synthetic Primer <4 OOs, SEQUENCE: 128 gggttt CCag cagcatttgt td 22

<210s, SEQ ID NO 129 &211s LENGTH: 24 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Synthetic Primer <4 OOs, SEQUENCE: 129 c cacttgatgtct coagcac taag 24

<210s, SEQ ID NO 130 &211s LENGTH: 26 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Synthetic Probe <4 OOs, SEQUENCE: 130 acat ct coat Ctgggacacc gcaggg 26

<210s, SEQ ID NO 131 &211s LENGTH: 21 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Synthetic Primer <4 OOs, SEQUENCE: 131 gcct tctacc talagcagtgg 21

<210s, SEQ ID NO 132 &211s LENGTH: 22 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Synthetic Primer <4 OOs, SEQUENCE: 132 tgc.cggtgat t cacat cata gg 22

<210s, SEQ ID NO 133 &211s LENGTH: 26 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Synthetic Probe US 2012/0070450 A1 Mar. 22, 2012 50

- Continued <4 OOs, SEQUENCE: 133 ttittgactico citggttgctic ccctgg 26

<210s, SEQ ID NO 134 &211s LENGTH: 23 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Synthetic Primer <4 OOs, SEQUENCE: 134 gccalacatca atgtggaaaa to 23

<210s, SEQ ID NO 135 &211s LENGTH: 2O &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Synthetic Primer <4 OOs, SEQUENCE: 135 ctgctic ct ct tctgctgg to 2O

<210s, SEQ ID NO 136 &211s LENGTH: 23 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Synthetic Probe <4 OOs, SEQUENCE: 136 cgc.cgt Cagg attgctg.ccg tca 23

<210s, SEQ ID NO 137 &211s LENGTH: 2O &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Synthetic Primer <4 OOs, SEQUENCE: 137 aggg accqct citcttct citc 2O

<210s, SEQ ID NO 138 &211s LENGTH: 2O &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Synthetic Primer <4 OOs, SEQUENCE: 138 cct caaccag ccagtgttcc 2O

<210s, SEQ ID NO 139 &211s LENGTH: 25 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Synthetic Probe <4 OOs, SEQUENCE: 139 agtict ccd.cg Ctgtagct co ttga 25 US 2012/0070450 A1 Mar. 22, 2012 51

- Continued

<210s, SEQ ID NO 140 &211s LENGTH: 2O &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Synthetic Primer <4 OOs, SEQUENCE: 140 c cct galaccc cagaacaacc

<210s, SEQ ID NO 141 &211s LENGTH: 23 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Synthetic Primer <4 OOs, SEQUENCE: 141 gggalagtgaa cagttittgga acc 23

<210s, SEQ ID NO 142 &211s LENGTH: 26 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Synthetic Probe < 4 OO > SEQUENCE: 142

CCttgtcggc tigtggtgtct Ctgctic 26

<210s, SEQ ID NO 143 &211s LENGTH: 21 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Synthetic Primer <4 OOs, SEQUENCE: 143 aagct ctittg ggctggtgat g 21

<210s, SEQ ID NO 144 &211s LENGTH: 24 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Synthetic Primer <4 OOs, SEQUENCE: 144 ggtggaagaa tt Cagaaga atgg 24

<210s, SEQ ID NO 145 &211s LENGTH: 28 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Synthetic Probe <4 OOs, SEQUENCE: 145 aaggactic cc acaacgaact caatcc.ca 28

<210s, SEQ ID NO 146 &211s LENGTH: 21 &212s. TYPE: DNA US 2012/0070450 A1 Mar. 22, 2012 52

- Continued <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Synthetic Primer <4 OOs, SEQUENCE: 146 tacgacatgc tiggggatcat g 21

<210s, SEQ ID NO 147 &211s LENGTH: 26 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Synthetic Primer <4 OOs, SEQUENCE: 147 gtagcc.gtaa acaacaatgg tatttic 26

<210s, SEQ ID NO 148 &211s LENGTH: 26 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Synthetic Probe <4 OOs, SEQUENCE: 148 tggit cogt cc acaa.gcaatgagtgcc 26

<210s, SEQ ID NO 149 &211s LENGTH: 21 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Synthetic Primer <4 OOs, SEQUENCE: 149 actgttggct gtgaggaatg C 21

<210s, SEQ ID NO 150 &211s LENGTH: 22 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Synthetic Primer <4 OOs, SEQUENCE: 150

CCttitt Caga gccttggagg ag 22

<210s, SEQ ID NO 151 &211s LENGTH: 25 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Synthetic Probe <4 OOs, SEQUENCE: 151 cc.cgagtgac aag cct gtag cc cat 25

<210s, SEQ ID NO 152 &211s LENGTH: 23 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Synthetic Primer US 2012/0070450 A1 Mar. 22, 2012 53

- Continued SEQUENCE: 152 c caggcagtic agat catctt citc 23

SEO ID NO 153 LENGTH: 2O TYPE: DNA ORGANISM: Artificial Sequence FEATURE: OTHER INFORMATION: Synthetic Primer

SEQUENCE: 153 citct cagotc. cacgc.cattg

SEQ ID NO 154 LENGTH: 24 TYPE: DNA ORGANISM: Artificial Sequence FEATURE: OTHER INFORMATION: Synthetic Probe

SEQUENCE: 154

CCttggctgg galagcacacc Ctgc 24

SEO ID NO 155 LENGTH: 18 TYPE: DNA ORGANISM: Artificial Sequence FEATURE: OTHER INFORMATION: Synthetic Primer

SEQUENCE: 155

Cctgcaa.gcc ctggacca 18

SEQ ID NO 156 LENGTH: 21 TYPE: DNA ORGANISM: Artificial Sequence FEATURE: OTHER INFORMATION: Synthetic Primer SEQUENCE: 156 agattgcgtc. c.gagct attg C 21

SEO ID NO 157 LENGTH: 25 TYPE: DNA ORGANISM: Artificial Sequence FEATURE: OTHER INFORMATION: Synthetic Probe SEQUENCE: 157 tcttctggac cctgaacggc acgct 25

SEO ID NO 158 LENGTH 19 TYPE: DNA ORGANISM: Artificial Sequence FEATURE: OTHER INFORMATION: Synthetic Primer SEQUENCE: 158 accagcticca ggagaaggc 19 US 2012/0070450 A1 Mar. 22, 2012 54

- Continued

SEO ID NO 159 LENGTH: 22 TYPE: DNA ORGANISM: Artificial Sequence FEATURE: OTHER INFORMATION: Synthetic Primer

SEQUENCE: 159

Cagttgcaag cagticttgag td 22

SEQ ID NO 160 LENGTH: 25 TYPE: DNA ORGANISM: Artificial Sequence FEATURE: OTHER INFORMATION: Synthetic Probe

SEQUENCE: 16 O citcct tactic gtccacgc.cg cctica 25

SEQ ID NO 161 LENGTH: 2O TYPE: DNA ORGANISM: Artificial Sequence FEATURE: OTHER INFORMATION: Synthetic Primer SEQUENCE: 161 tcc.gagaagt ggttgctgac

SEQ ID NO 162 LENGTH: 26 TYPE: DNA ORGANISM: Artificial Sequence FEATURE: OTHER INFORMATION: Synthetic Primer SEQUENCE: 162 t cittgaaata acccaaatgt gaatcc 26

SEQ ID NO 163 LENGTH: 26 TYPE: DNA ORGANISM: Artificial Sequence FEATURE: OTHER INFORMATION: Synthetic Probe SEQUENCE: 163 cgctgtagac atc.cgacctic tdaccc 26

SEQ ID NO 164 LENGTH: 21 TYPE: DNA ORGANISM: Artificial Sequence FEATURE: OTHER INFORMATION: Synthetic Primer SEQUENCE: 164

Ctgaga.ccga gtc.gc.ctt at C 21

SEQ ID NO 165 LENGTH: 21 TYPE: DNA US 2012/0070450 A1 Mar. 22, 2012 55

- Continued <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Synthetic Primer <4 OOs, SEQUENCE: 165 atacggcctic ttgttgttga g 21

<210s, SEQ ID NO 166 &211s LENGTH: 27 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Synthetic Probe <4 OOs, SEQUENCE: 166 agtaccgata acago catgc actgtgc 27

<210s, SEQ ID NO 167 &211s LENGTH: 24 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Synthetic Primer <4 OOs, SEQUENCE: 167 agtgctgttga tigatggacaa ttac 24

<210s, SEQ ID NO 168 &211s LENGTH: 2O &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Synthetic Primer <4 OOs, SEQUENCE: 168 ttgctict cct gtgcagaagg

<210s, SEQ ID NO 169 &211s LENGTH: 26 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Synthetic Probe <4 OOs, SEQUENCE: 169 t ct caccagt gtgct tcc td citgtgc 26

<210s, SEQ ID NO 170 &211s LENGTH: 2O &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Synthetic Primer <4 OOs, SEQUENCE: 170 gtcggCatct gagaccagtg

<210s, SEQ ID NO 171 &211s LENGTH: 23 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Synthetic Primer US 2012/0070450 A1 Mar. 22, 2012 56

- Continued <4 OOs, SEQUENCE: 171 gttcacagtic ctitgaagttca cac 23

<210s, SEQ ID NO 172 &211s LENGTH: 26 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Synthetic Probe <4 OOs, SEQUENCE: 172

C Cactggttctggactgctic ticgtt 26

<210s, SEQ ID NO 173 &211s LENGTH: 21 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Synthetic Primer <4 OOs, SEQUENCE: 173

Cccagaggaa acggacacaa C 21

<210s, SEQ ID NO 174 &211s LENGTH: 23 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Synthetic Primer <4 OOs, SEQUENCE: 174 tgcagatgct tct cotgttg tag 23

<210s, SEQ ID NO 175 &211s LENGTH: 14 O &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: PCR product <4 OO > SEQUENCE: 175 gtgctttctg gagggit ctac tatgt atcct ggcctgc.cat Cacggttgga acgagaactt 6 O aaac agctitt acttagaacg agttittgaag gigtigatgtgg aaaaactitt c taaatttaag 12 O atcc.gcattg aag acccacc 14 O

<210s, SEQ ID NO 176 &211s LENGTH: 150 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: PCR product <4 OOs, SEQUENCE: 176 gtagagtgga aaaggagc at tatatgat gat actgatg agt cc cactg. tctgagcac 6 O attgagt cac gtact cittgc aattgc.ccgc aacctgactic agcagct coa gaccacgtgc 12 O cacaccct co tdtccaa.cat coaaggtgta 15 O

<210s, SEQ ID NO 177 &211s LENGTH: 108 &212s. TYPE: DNA US 2012/0070450 A1 Mar. 22, 2012 57

- Continued <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: PCR product <4 OO > SEQUENCE: 177 gctgct coat tittaaata citt CC aggala aaggggotca toatgacatt tatgc.cgac 6 O cgcgatgagg atgaggtgtt Ctatgacatc agcatggcag ttgacaac 108

<210s, SEQ ID NO 178 &211s LENGTH: 116 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: PCR product <4 OOs, SEQUENCE: 178 agtactttgt C9gttacct a ggaga.gagaa cqcagagcac ccctggctac at atttggga 6 O aacgcatcat act citt cotg titcct catgt ccgttgctgg catatt caac tattac 116

<210s, SEQ ID NO 179 &211s LENGTH: 124 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: PCR product < 400 SEQUENCE: 179 gct cagtgtg gagtat ct ca aagcc titt ca ggctgtc.cag aatct tccala C caagaa.gca 6 O gcaact acag gotttgaacc ttcttgtcat cotcc tacct gatgcaaac a gggacacact 12 O galag 124

<210s, SEQ ID NO 18O &211s LENGTH: 103 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: PCR product <4 OOs, SEQUENCE: 18O tcggggacga gatggacgtg agcct caggg ccc.cgc.gc.ct ggcc.cagctic ticcgaggtgg 6 O c catgcacag cctgggtctg gctitt Catct acgaccagac ta 103

<210s, SEQ ID NO 181 &211s LENGTH: 145 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: PCR product <4 OOs, SEQUENCE: 181 ccacagcaat ttctgggitta cagagt cagc gaatgaggca cagcaac cac to Cacagaga 6 O actic to caat tdaaagacga agtictaatga cct ctdatga aaattat cac aatgaaaggg 12 O

Ctcggaagag taggalacc.gc titgtc. 145

<210s, SEQ ID NO 182 &211s LENGTH: 109 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: US 2012/0070450 A1 Mar. 22, 2012 58

- Continued <223> OTHER INFORMATION: PCR product <4 OOs, SEQUENCE: 182 t caaggagta tittctacacc agtggcaagt gct coaacco agcagt cq t c tttgtcaccc 6 O gaaagaaccg C caagtgtgt gccaa.cccag agaagaaatg ggttcggga 109

<210s, SEQ ID NO 183 &211s LENGTH: 136 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: PCR product <4 OOs, SEQUENCE: 183

Cagcagtggg Caggaatgac acccacccta C cacagggtc agcct coccg aaa.caccaga 6 O agaagt cc aa gttacatggc cccactgaaa cct caagctg ttcaggtgcc gcc cc tactg 12 O tggagatgga tgagga 136

<210s, SEQ ID NO 184 &211s LENGTH: 148 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: PCR product < 4 OO > SEQUENCE: 184 tgg tactggc taccct tctic ticgcaagtga gcc ccttcaa gatacctata gaggaacttg 6 O aggacagagt gtttgttgaat tcaatacca gcatcacatg ggtagaggga acggtgggaa 12 O cactgct citc agacattaca agactgga 148

<210s, SEQ ID NO 185 &211s LENGTH: 132 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: PCR product <4 OOs, SEQUENCE: 185 aatgcggcag acagtt actic ctgggttcca gagcgagctg agagc agggc catggagaac 6 O

Cagtacagtic cacacctgg gacagactgc tigaaagtgag gtggc cc tag agaCact aga 12 O gtcaccagcc ac 132

<210s, SEQ ID NO 186 &211s LENGTH: 99 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: PCR product <4 OOs, SEQUENCE: 186 citatgtgttt accatcctica actgcctgca ggg.cgcc titc citctacctgc tigcactgcct 6 O gct Caacaag aaggttcggg aagaataccg gaagtgggc 99

<210s, SEQ ID NO 187 &211s LENGTH: 143 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: US 2012/0070450 A1 Mar. 22, 2012 59

- Continued <223> OTHER INFORMATION: PCR product <4 OOs, SEQUENCE: 187 acatgaatat citccaacaag at caggaacc tict coaccac actgcaaaca atagocacca 6 O aattatgtcg tagdt at at agcaaagaac aagagcacaa atgtaagcct tdt CCaagga 12 O gatggatttg gCataaggac agc 143

<210s, SEQ ID NO 188 &211s LENGTH: 128 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: PCR product <4 OOs, SEQUENCE: 188 tctt coaggt gggctic cagc ggttcc.ca.gc gcgatgtcala Ctgct cq9tt atgggaccac 6 O aagaaaaaaa agtagtggtg tacct tcaga agctggatac agcatatgat gaccttggca 12 O attctggc 128

<210s, SEQ ID NO 189 &211s LENGTH: 150 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: PCR product <4 OOs, SEQUENCE: 189 tgat cott at t cagtag cact citctgaact tdgagacagg caaaatgaaa gttat ct caa 6 O ttittgctcga aagagaataa agaac cct ga aggaggcctg tacctggctg taccCtgtt 12 O ggcaggcatc actggagttgtcatcacgct 15 O

<210s, SEQ ID NO 190 &211s LENGTH: 121 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: PCR product <4 OOs, SEQUENCE: 190 agctgtacga Ctggcc ctac aggtttctgc ggcgctittgg gcgggacaag gta acct titt 6 O

CCtttgaggc aggc.cgt.cgc tigcgt.ctctg gagagggcaa Ctttgagttc gaalacc.cggc 12 O a. 121

<210s, SEQ ID NO 191 &211s LENGTH: 87 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: PCR product <4 OOs, SEQUENCE: 191 gagaaatcag atggtgttta cacgggcctg agcaccagga accaggagaci ttacgagact 6 O

Ctgaag catg agaalaccacc acagtag 87

<210s, SEQ ID NO 192 &211s LENGTH: 137 &212s. TYPE: DNA US 2012/0070450 A1 Mar. 22, 2012 60

- Continued <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: PCR product <4 OOs, SEQUENCE: 192 gatgactatg gagaccCaaa ttct Cagaa totatgtc.cc agaalacctgt ggctgct tca 6 O accattgaca gttittgctgc tigctggcttctgcagacagt caa.gctgcag ctic ccc.caaa 12 O ggctgtgctgaaacttg 137

<210s, SEQ ID NO 193 &211s LENGTH: 110 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: PCR product <4 OOs, SEQUENCE: 193 titt cittaaat gg.ccca catc aggacagotg titccttggcc atcc.caaagc tatt ctdggg 6 O gcaa.ca.gagg taalactact gggcc atgga cagcc actta actggattt C 11 O

<210s, SEQ ID NO 194 &211s LENGTH: 147 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: PCR product <4 OOs, SEQUENCE: 194 accaccitcca ccatcc catc cqgc.cago.ca accaccattg ccago at ct c acc catcaca 6 O accaccagtic ccaa.gc.ctac ctic ccagaaa cattaaacct c cqtttgacc taaaaag.ccc 12 O tgtcaatgaa gacaat Caag atggtgt 147

<210s, SEQ ID NO 195 &211s LENGTH: 135 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: PCR product <4 OOs, SEQUENCE: 195 cataccataa caatgacgac aactt cagtic agcagotggc ctitactic ct c ccacagaatg 6 O cgctittataa ccaat catag cqaccaac cq ccacaaaact tct cagdaac accaaatgtt 12 O actacctgtc. c catg 135

<210s, SEQ ID NO 196 &211s LENGTH: 144 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: PCR product <4 OOs, SEQUENCE: 196

Ctttgggtga gttgaact tc ttic cattata gaaagaattg aaggctgaga aacticagoct 6 O citat catgtg gaacagotct gacgc.caact tct cotgcta ccatgagtct gtgctgggct 12 O atcgittatgt to agittagc tiggg 144

<210s, SEQ ID NO 197 US 2012/0070450 A1 Mar. 22, 2012 61

- Continued

&211s LENGTH: 84 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: PCR product <4 OO > SEQUENCE: 197 c cct tcctac toccagacac ccaccct cqc titcagocaca gtttcct cat citgtc.ca.gtg 6 O gg taggttgg actggaaaat Ctct 84

<210s, SEQ ID NO 198 &211s LENGTH: 98 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: PCR product <4 OOs, SEQUENCE: 198 gatc catctg gigt cacatcc tict tcctgct tttgctic cca gtggctgcag ct cagacgac 6 O tccaggagag agat catcac tocct gcc.tt ttaccctg 98

<210s, SEQ ID NO 199 &211s LENGTH: 114 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: PCR product <4 OOs, SEQUENCE: 199 gccatgaatt tacct citat gaaaacaaag act acattag aaactgcatc attgg taaag 6 O gacgcagcta Caagggalaca gitatictatica Ctalagagtgg catcaaatgt cagc 114

<210s, SEQ ID NO 2 OO &211s LENGTH: 79 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: PCR product <4 OOs, SEQUENCE: 2OO cagttcagca toagttccaa gacacgtttc caggit cocta togctgtgctic acgaaggaca 6 O c catgcc.gca gacgtacaa 79

<210s, SEQ ID NO 2 O1 &211s LENGTH: 110 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: PCR product <4 OOs, SEQUENCE: 2O1 aaactgttcc gcagdcagag cqctgatgcc ccc.ggc.ccca cagagcgcga gcggctaaag 6 O aagatgttgt Ctgagggctic cqtgggcgag gtacagtggg aggcc.gagtt 11 O

<210s, SEQ ID NO 202 &211s LENGTH: 104 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: PCR product US 2012/0070450 A1 Mar. 22, 2012 62

- Continued <4 OOs, SEQUENCE: 2O2

Cctagaggat Cttgtt actgaataccacgg gaacttitt.cg gcctggagtg gtgttgtctaa 6 O gggactggct gagagt ctgc agc.ca.gacta Cagtgaacga Citct 104

<210s, SEQ ID NO 203 &211s LENGTH: 104 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: PCR product <4 OOs, SEQUENCE: 2O3 cc.cgcatc cc ticacatgaaa gaccc catcg gtgacagctt C caaaacgac aagctggtgg 6 O tctgggaggc gggcaaagcc ggcctggagg agtgtctggit gact 104

<210s, SEQ ID NO 204 &211s LENGTH: 150 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: PCR product <4 OOs, SEQUENCE: 204 t cct gctggit Catctggaag gctctgat Co acctgagcga cct Cogggag tacaggcgct 6 O ttgaga agga gaagct caag ticc cagtgga acaatgataa t cc ccttitt C aagagcgc.ca 12 O ccacgacggt catgalacc cc aagtttgctg 15 O

<210s, SEQ ID NO 205 &211s LENGTH: 127 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: PCR product <4 OOs, SEQUENCE: 205 gcgggaggct gtc.ttt CCct tccagcctgg aagtgttgca gaggtgtgca t caccitt.cga 6 O c caggccaac ctdaccgt.ca agctgccaga tiggatacgaa ttcaagttcc cca accqcct 12 O caacct g 127

<210s, SEQ ID NO 206 &211s LENGTH: 150 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: PCR product <4 OOs, SEQUENCE: 2O6 tgaag.cccaa gagccaaagg aat caccacc acctt ctaaa acgtcagcag ctgct cagtt 6 O ggatgagctic atggct cacc tactgagat gcaggccaag gttgcagtga gag cagatgc 12 O tggcaagaag cacttaccag acaag cagga 15 O

<210s, SEQ ID NO 2 O7 &211s LENGTH: 85 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: PCR product US 2012/0070450 A1 Mar. 22, 2012 63

- Continued <4 OOs, SEQUENCE: 2O7

CCttgacctt ggggagaacc agttggagac Cttgccacct gacct Cotgaggggit cogct 6 O gcaattagaa cqgcta catc tagaa 85

<210s, SEQ ID NO 208 &211s LENGTH: 118 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: PCR product <4 OOs, SEQUENCE: 208 atgtct caag gct catctgt tttgaattitc. tcc tatcc.ca totgtgaggc ggctctg.ccc 6 O aagttittctt totgtggaag aaggaaagga gag cagattt act atgctgg gcctgtca 118

<210s, SEQ ID NO 209 &211s LENGTH: 116 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: PCR product <4 OOs, SEQUENCE: 209 atatt catgt tttacaagga gaalacccatt gattggcticc toggaccatat t citctgggtg 6 O aaagtctgca acc ctgaaaa agatgcaaaa cattgtgata gacagaaag.c aaatct 116

<210s, SEQ ID NO 210 &211s LENGTH: 133 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: PCR product <4 OOs, SEQUENCE: 210 agagcgtgtc. cccacagggc aac agcgttg accgcatggc agctc.cgaga gcagaggctic 6 O tatttgactt Cactggaaac agcaaactgg agctgaattt Caaagctgga gatgtgatct 12 O t cct cotcag tog 133

<210s, SEQ ID NO 211 &211s LENGTH: 87 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: PCR product <4 OOs, SEQUENCE: 211 ggggagcact act cog agaia gttacgagaa citggt cagca totgcatctg C cctgacccC 6 O

Caccagagac Ctgacatcgg atacgtg 87

<210s, SEQ ID NO 212 &211s LENGTH: 125 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: PCR product <4 OOs, SEQUENCE: 212 agaggittata atctgaatcc caaaggagac to agctgat gaaagtgctt C caaactgaa 6 O US 2012/0070450 A1 Mar. 22, 2012 64

- Continued aattggacgt gcc tittacga tigg taag.cgt taa.ca.gcticc cactgcttct ataatgactic 12 O citt ta. 125

<210s, SEQ ID NO 213 &211s LENGTH: 142 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: PCR product <4 OOs, SEQUENCE: 213 gact acagca acgaggagca catgaccctg. Ctgaagatga titttgataaa atgctgttgat 6 O atct ctaacg aggtocgt.cc aatggaagtic gcagagcctt gggtggactg. titt attagag 12 O gaat attitta to agagcga cc 142

<210s, SEQ ID NO 214 &211s LENGTH: 81 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: PCR product <4 OOs, SEQUENCE: 214 acatgt attic aactgcacca agacct citg ttgagacctic cc.gc.gcagtg cct Ctggctg 6 O gttittggitta tdgattgc cc a 81

<210s, SEQ ID NO 215 &211s LENGTH: 125 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: PCR product <4 OOs, SEQUENCE: 215 gatcagactic agcctic ct ca gag cctgttg ggatatat ca gggtttcgag aagaagaccg 6 O gagttgctgg ggagga catg Caaga caa.ca gtgggaccta C9gcaagat C tdgagggca 12 O gcagc 125

<210s, SEQ ID NO 216 &211s LENGTH: 105 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: PCR product <4 OOs, SEQUENCE: 216 tgg.cccagag toggcatgtcc cagccagacc cct cotggcc gct actic titt titc cct citt c 6 O tctgggct ct cccact cotgggg.ccggit ct gag cctacct gag cc 105

<210s, SEQ ID NO 217 &211s LENGTH: 96 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: PCR product <4 OOs, SEQUENCE: 217 gggttt CCag cagcatttgt to agcatgg gctgtggcac gagcaactict ggctgatgcg 6 O US 2012/0070450 A1 Mar. 22, 2012 65

- Continued aggtgctggg aacttagtgc tiggaga catc aagtgg 96

<210s, SEQ ID NO 218 &211s LENGTH: 14 O &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: PCR product <4 OOs, SEQUENCE: 218 gcct tctacc talagcagtg gcgct Cotac alacat ct coa tictgggacac cqc agggcgg 6 O gaggagttcc acggcctggg Ctic catgitac to cqggggg C9gcc.gc.cat catcct Cacc 12 O tatgatgtga at Caccggca 14 O

<210s, SEQ ID NO 219 &211s LENGTH: 14 O &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: PCR product <4 OOs, SEQUENCE: 219 gccaa.catca atgtggaaaa togcatttitt.c act ct cqcca gagatat caa agcaaaaatg 6 O gacaaaaaat taaggcaa cagcc.cccag gggagcaa.cc agggagt caa aat cacaccg 12 O gacCagcaga agaggagcag 14 O

<210s, SEQ ID NO 220 &211s LENGTH: 119 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: PCR product <4 OOs, SEQUENCE: 220 aggg accqct citcttctic to go.cga-cagct titt cott coc to catgagaa agaa.gc.cagc 6 O tctgtctgac ggcagcaatc Ctgacggcga t ct cotc.cag galacactggc tiggttgagg 119

<210s, SEQ ID NO 221 &211s LENGTH: 91 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: PCR product <4 OOs, SEQUENCE: 221

CCCtgaac cc Cagaacaa.cc agctggat.ca gttct cacag gagct acagc gcggagactg 6 O ggaaacatgg titccaaaact gttcact tcc c 91

<210s, SEQ ID NO 222 &211s LENGTH: 148 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: PCR product <4 OOs, SEQUENCE: 222 aagct ctittg ggctggtgat ggccttgtcg gctgtggtgt Ctctgct coa gttc.cccatc 6 O ttcaccct catcaaaggctic cct tcagaat gacccatttit acgtgaatgt gatgttcatg 12 O US 2012/0070450 A1 Mar. 22, 2012 66

- Continued cittgcc attc ttctgacatt citt coacc 148

<210s, SEQ ID NO 223 &211s LENGTH: 150 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: PCR product <4 OOs, SEQUENCE: 223 tacgacatgc tiggggatcat ggcatggaat gcaacttgca aaaactggct ggcagcagag 6 O gctg.ccctgg aaaagtacta Cctitt CCatt ttt tatggga ttgagttcgt ttgggagtic 12 O cittggaaata ccattgttgt ttacggctac 15 O

<210s, SEQ ID NO 224 &211s LENGTH: 112 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: PCR product <4 OOs, SEQUENCE: 224 actgttggct gtgaggaatg cacagtgttt coctdttitat ccatc.ccct g caaactgcag 6 O agtggc actic attgcttgttg gacggaccag Ctic ct coaag gct ctgaaaa gg 112

<210s, SEQ ID NO 225 &211s LENGTH: 14 O &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: PCR product <4 OOs, SEQUENCE: 225 cCaggcagtic agatcatctt Ctcgalacc cc gagtgacaag cct gtagcc C atgttgtagc 6 O aaac cct caa gctgaggggc agctic cagtg gctgaac.cgc C9gc caatig C cct cctggc 12 O

Caatggcgtg gagctgaga.g 14 O

<210s, SEQ ID NO 226 &211s LENGTH: 78 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: PCR product <4 OOs, SEQUENCE: 226

Cctgcaa.gcc ctggaccaac to accttgg Ctgggaa.gca Caccctgcag ccggc.ca.gca 6 O at agct cqga cqcaat ct 78

<210s, SEQ ID NO 227 &211s LENGTH: 97 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: PCR product <4 OOs, SEQUENCE: 227 accagcticca ggagaaggca act coagt ca galacagoaga aataagcgt.g. cc.gttcaggg 6 O tccagaagaa acagt cactic aag actgctt gcaactg 97 US 2012/0070450 A1 Mar. 22, 2012 67

- Continued

<210s, SEQ ID NO 228 &211s LENGTH: 1.OO &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: PCR product <4 OOs, SEQUENCE: 228 tcc.gagaagt ggttgctgac agccacaaag taaagggag taggcggcg tdgacgagta 6 O aggagtgaca gtgaggattic acatttgggit tatttcaaga

<210s, SEQ ID NO 229 &211s LENGTH: 8O &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: PCR product <4 OOs, SEQUENCE: 229

Ctgaga.ccga gtc.gc.ctt at Caggagct Co agggit cagag gtcggatgtc. tacagcgacc 6 O t caacacaca gaggc.cgitat

<210s, SEQ ID NO 230 &211s LENGTH: 82 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: PCR product <4 OOs, SEQUENCE: 23 O agtgctgttga tigatggacaa ttacatagta C catalacag C catgcactg. tcaaag cat 6 O gccCttctgc acaggaga.gc aa 82

<210s, SEQ ID NO 231 &211s LENGTH: 149 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: PCR product <4 OOs, SEQUENCE: 231 gtcggCatct gagaccagtg agaaacgc.cc Ctt catgtgt gcttacc cag gctgcaataa 6 O gagatattitt aagctgtc.cc acttacagat gcacagc agg aag cacactg gtgagaalacc 12 O ataccagtgt gaCttcaagg actgttgaac 149

<210s, SEQ ID NO 232 &211s LENGTH: 124 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: PCR product

<4 OOs, SEQUENCE: 232

Cccagaggaa acggacacaa citc.cgggaag cct ttgagca gct coaggcc aagaaacaaa 6 O tggc catgga gaaacgcaga gcagt ccaga accagtggca gct acaa.cag gagaa.gcatc 12 O tgca 124

US 2012/0070450 A1 Mar. 22, 2012 69

TESC, THEX1, TMEM30A, TMEM33, TRIP13, CALCRL, CCL4, CCL5, CD33, CD36, CD3D, CD86, TUBB6, UBASH3B, UGCG, VSTM1, WDR4, WIT1, CD9, CD93, CD96, CD97, CFD, CHI3L1, CLEC12A, WSB2 and ZNF253; CLECL1, COCH, CST7, CXCL1, DOK2, EMR2, or a Substance capable of Suppressing the activity of a FCER1G, FCGR2A, FUCA2, GPR109B, GPR160, translation product of the gene. GPR34, GPR84, HAVCR2, HBEGF, HCST, HGF, 4. The therapeutic agent according to claim 3, wherein the HLA-DOB, HOMER3, IFI30, IL13RA1, IL2RA, leukemic stem cell marker gene is selected from the group IL2RG, IL3RA, INHEA, ITGB2, LGALS1, LRG1, consisting of: LY86, MAMDC2, MGAT4A, P2RY14, P2RY5, cell membrane- or extracellularly-localized genes consist PLAUR, PPBP, PRG2, PRSS21, PTH2R, PTX3, ing of ADFP. ALOX5AP CACNB4, CCL5, CD33, REEP5, RNASE2, RXFP1, SLC31A2, SLC43 A3, CD3D, CD93, CD97, CLEC12A, DOK2, FCER1G, SLC6A6, SLC7A6, STX7, SUCNR1, TACSTD2, FCGR2A, FUCA2, GPR34, GPR84, HCST, HGF, TIMP1, TM4SF1, TM9SF1, TNF, TNFRSF4, HOMER3, IL2RA, IL2RG, IL3RA, ITGB2, LGALS1, TNFSF13B, TYROBP, UTS2 and VNN1; LRG1, LY86, MGAT4A, P2RY5, PRSS21, PTH2R, cell cycle-related genes consisting of AURKA, C13orf34, RNASE2, SLC43A3, SUCNR1, TIMP1, TNF, CCNA1, DSCC1, FAM33A, HPGD, NEK6, PYHIN1, TNFRSF4, TNFSF13B, TYROBP and VNN1; cell RASSF4, TXNL4B and ZWINT: cycle-related genes consisting of ZWINT, NEK6 and apoptosis-related genes consisting of MPO, IER3, BIK, TXNL4B; an apoptosis-related gene consisting of BIK; TXNDC1, GADD45B and NAIP; signaling-related genes consisting of AK5. signaling-related genes consisting of AK5, ARHGAP18. ARHGAP18, FYB, HCK, LPXN, PDE9A, PDK1, ARRB1, DUSP6, FYB, HCK, LPXN, MS4A3, PRKCD, RAB20, RAB8A and RABIF; transcription PAK1IP1, PDE9A, PDK1, PRKAR1A, PRKCD, PXK, factor genes consisting of WT1 and HLX; and other RAB20, RAB8A, RABIF, RASGRP3, RGS18 and genes consisting of CYBB, CTSC and NCF4. S100A11; 5. The therapeutic agent according to claim 3, wherein the transcription factor genes consisting of WT1, MYC and leukemic stem cell marker gene is selected from the group HLX; and consisting of: other genes consisting of ACTR2. ALOX5, ANXA2P2, cell membrane- or extracellularly-localized genes consist ATL3, ATP6V1B2, ATP6V1C1, ATP6V1D, C12orf5, ing of ALOX5AP CACNB4, CCL5, CD33, CD3D, C17orf60, C18 orf19, C1GALT1C1, C1orf135, CD93, CD97, CLEC12A, DOK2, FCGR2A, GPR84, C1orf163, Clorf186, C60rf150, CALML4, CCT5, HCST, HOMER3, ITGB2, LGALS1, LRG1, PTH2R, CLC, COMMD8, COTL1, COX17, CRIP1, CSTA, RNASE2, TNF, TNFSF13B, TYROBP and VNN1; a CTSA, CTSC, CTSG, CYBB, CYP2E1, DENND3, cell cycle-related gene consisting of NEK6; an apopto DHRS3, DLAT, DLEU2, DPH3, EFHD2, ENC1, sis-related gene consisting of BIK, signaling-related EXOSC3, FAM107B, FAM129A, FAM38B, FBXO22, genes consisting of AK5, FYB, HCK, LPXN, PDE9A, FLJ14213, FNDC3B, GNPDA1, GRPEL1, GTSF1, PDK1, PRKCD and RAB20; a transcription factor gene HIG2, HN1, HVCN1, IDH1, IDH3A, IKIP, KIF2C, consisting of WT1; and other genes consisting of CTSC KYNU, LCMT2, ME1, MIRN21, MKKS, MNDA, and NCF4. MTHFD2, MYO1B, MYO1F, NAGA, NCF2, NCF4, 6. The therapeutic agent according to claim 3, wherein the NDUFAF1, NP, NRIP3, OBFC2A, PARP8, PDLIM1, leukemic stem cell marker gene is a marker expressed in stem PDSS1, PGM2, PIGK, PIWIL4, PPCDC, PPIF, cells that are present in bone marrow niches, are in the sta PRAME, PUS7, RPP40, RRM2, S100A16, S100A8, tionary phase of cell cycle, and are resistant to anticancer SLOOP, S100Z, SAMHD1, SH2D1A, SPCS2, agents, selected from the group consisting of AK5, BIK, SPPL2A, TESC, THEX1, TMEM30A, TMEM33, DOK2, FCGR2A, IL2RA, LRG1, SUCNR1 and WT1. TRIP13, TUBB6, UBASH3B, UGCG, VSTM1, WDR4, 7. The therapeutic agent according to claim 3, wherein the WIT1, WSB2 and ZNF253; and Substance capable of suppressing the expression of the gene is c) a step of sorting cells to which the Substance has bound, an antisense nucleic acid oran RNAi-inducible nucleic acid. and obtaining the sample from which leukemic stem 8. The therapeutic agent according to claim 3, wherein the cells have been purged. Substance capable of Suppressing the activity of the transla 11. The production method according to claim 10, wherein tion product is an aptamer or an antibody. the leukemic stem cell marker is at least one kind of cell 9. The therapeutic agent according to claim 8, wherein the surface marker gene selected from among ADFP. ALOX5AP, antibody is an immunoconjugate of an antibody and an anti CACNB4, CD33, CD3D, CD93, CD97, CLEC12A, DOK2, cancer Substance. FCER1G, FCGR2A, GPR34, GPR84, HCST, HOMER3, 10. A production method of a sample containing hemato IL2RA, IL2RG, IL3RA, ITGB2, LY86, P2RY5, PTH2R, poietic cells for autologous transplantation or allogeneic SUCNR1, TNFRSF4, TYROBP and VNN1. transplantation for a patient with acute myeloid leukemia, 12. A method for preventing or treating acute myeloid comprising: leukemia that targets leukemic stern cells, comprising admin a) a step of collecting a sample containing hematopoietic istering, to a subject, an effective amount of a Substance cells from the patient or a donor; capable of suppressing the expression of a gene selected from b) a step of bringing the collected sample into contact with among leukemic stern cell marker genes consisting of the a Substance that recognizes a translation product of at following set of genes: least one kind of leukemic stem cell marker gene cell membrane- or extracellularly-localized genes consist Selected from among the following set of genes: ing of ADFP. ALOX5AP, AZU1, C3AR1, CACNB4, cell membrane- or extracellularly-localized genes consist CALCRL, CCL4, CCL5, CD33, CD36, CD3D, CD86, ing of ADFP. ALOX5AP, AZU1, C3AR1, CACNB4, CD9, CD93, CD96, CD97, CFD, CHI3L1, CLEC12A, US 2012/0070450 A1 Mar. 22, 2012 70

CLECL1, COCH, CST7, CXCL1, DOK2, EMR2, PDSS1, PGM2, PIGK, PIWIL4, PPCDC, PPIF, FCER1G, FCGR2A, FUCA2, GPR109B, GPR160, PRAME, PUS7, RPP40, RRM2, S100A16, S100A8, GPR34, GPR84, HAVCR2, HBEGF, HCST, HGF, S100P, S100Z, SAMHD1, SH2D1A, SPCS2, SPPL2A, HLA-DOB, HOMER3, IFI30, IL13RA1, IL2RA, TESC, THEX1, TMEM30A, TMEM33, TRIP13, IL2RG, IL3RA, INHEA, ITGB2, LGALS1, LRG1, TUBB6, UBASH3B, UGCG, VSTM1, WDR4, WIT1, LY86, MAMDC2, MGAT4A, P2RY14, P2RY5, WSB2 and ZNF253; or a substance capable of Suppressing the activity of a PLAUR, PPBP, PRG2, PRSS21, PTH2R, PTX3, translation product of the gene. REEP5, RNASE2, RXFP1, SLC31A2, SLC43 A3, 13. The therapeutic agent according to claim 4, wherein the SLC6A6, SLC7A6, STX7, SUCNR1, TACSTD2, Substance capable of suppressing the expression of the gene is TIMP1, TM4SF1, TM9SF1, TNF, TNFRSF4, an antisense nucleic acid oran RNAi-inducible nucleic acid. TNFSF13B, TYROBP, UTS2 and VNN1; 14. The therapeutic agent according to claim 4, wherein the cell cycle-related genes consisting of AURKA, C13orf34, Substance capable of Suppressing the activity of the transla CCNA1, DSCC1, FAM33A, HPGD, NEK6, PYHIN1, tion product is an aptamer or an antibody. RASSF4, TXNL4B and ZWINT: 15. The therapeutic agent according to claim 14, wherein apoptosis-related genes consisting of MPO, IER3, BIK, the antibody is an immunoconjugate of an antibody and an TXNDC1, GADD45B and NAIP; anticancer Substance. signaling-related genes consisting of AK5, ARHGAP18. 16. The therapeutic agent according to claim 5, wherein the ARRB1, DUSP6, FYB, HCK, LPXN, MS4A3, Substance capable of suppressing the expression of the gene is PAK1IP1, PDE9A, PDK1, PRKAR1A, PRKCD, PXK, an antisense nucleic acid oran RNAi-inducible nucleic acid. RAB20, RAB8A, RABIF, RASGRP3, RGS18 and 17. The therapeutic agent according to claim 5, wherein the S100A11; Substance capable of Suppressing the activity of the transla transcription factor genes consisting of WT1, MYC and tion product is an aptamer or an antibody. HLX; and 18. The therapeutic agent according to claim 17, wherein other genes consisting of ACTR2. ALOX5, ANXA2P2, the antibody is an immunoconjugate of an antibody and an ATL3, ATP6V1B2, ATP6V1C1, ATP6V1D, C12orf5, anticancer Substance. C7orf60, C18orf19, C1GALT1C1, C1orf135, 19. The therapeutic agent according to claim 6, wherein the C1orf163, C1orf186, C60rf150, CALML4, CCT5, Substance capable of suppressing the expression of the gene is CLC, COMMD8, COTL1, COX17, CRIP1, CSTA, an antisense nucleic acid oran RNAi-inducible nucleic acid. CTSA, CTSC, CTSG, CYBB, CYP2E1, DENND3, 20. The therapeutic agent according to claim 6, wherein the DHRS3, DLAT, DLEU2, DPH3, EFHD2, ENC1, Substance capable of Suppressing the activity of the transla EXOSC3, FAM107B, FAM129A, FAM38B, FBXO22, tion product is an aptamer or an antibody. FLJ14213, FNDC3B, GNPDA1, GRPEL1, GTSF1, 21. The therapeutic agent according to claim 20, wherein HIG2, HN1, HVCN1, IDH1, IDH3A, IKIP, KIF2C, the antibody is an immunoconjugate of an antibody and an KYNU, LCMT2, ME1, MIRN21, MKKS, MNDA, anticancer Substance. MTHFD2, MYO1B, MYO1F, NAGA, NCF2, NCF4, NDUFAF1, NP, NRIP3, OBFC2A, PARP8, PDLIM1,