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WO2007037611A1 - Marqueurs pour predire la reponse d'un patient souffrant de leucemie aigue myeloide a des medicaments anticancereux - Google Patents

Marqueurs pour predire la reponse d'un patient souffrant de leucemie aigue myeloide a des medicaments anticancereux Download PDF

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Publication number
WO2007037611A1
WO2007037611A1 PCT/KR2006/003822 KR2006003822W WO2007037611A1 WO 2007037611 A1 WO2007037611 A1 WO 2007037611A1 KR 2006003822 W KR2006003822 W KR 2006003822W WO 2007037611 A1 WO2007037611 A1 WO 2007037611A1
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Prior art keywords
response
predicting
patients
gene
aml
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Ceased
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PCT/KR2006/003822
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English (en)
Inventor
Jeong Ho Yoon
Young Joon Kim
Se Nyun Kim
Young-Hwa Song
Dong Yoon Park
Sung Han Kim
Han Yong Lee
Dong-Wook Kim
Inkyung Shin
Bong Yong Lee
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Daewoong Co Ltd
Digital Genomics Inc
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Daewoong Co Ltd
Digital Genomics Inc
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Priority to CN200680044375.1A priority Critical patent/CN101326290B/zh
Priority to JP2008533238A priority patent/JP5161091B2/ja
Publication of WO2007037611A1 publication Critical patent/WO2007037611A1/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C12Q1/6886—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/106—Pharmacogenomics, i.e. genetic variability in individual responses to drugs and drug metabolism
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/118—Prognosis of disease development
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/158—Expression markers

Definitions

  • the present invention relates to a marker for predicting the response of a patient with acute myeloid leukemia (AML) to anti-cancer drugs. More particularly, the present invention relates to a kit for predicting the response of AML patients to anticancer drugs, which comprises an agent for measuring the expression level of the marker, and a method for predicting the response of AML patients to anticancer drugs on the basis of the extent of expression of the marker.
  • AML acute myeloid leukemia
  • Leukemia may be classified into acute and chronic forms according to the progression rate thereof.
  • the clinic conditions of leukemia are various according to disease type and the chracters of the affected cells.
  • leukemia affects the lymphoid cells, it is called lymphocytic leukemia.
  • myeloid leukemia When myeloid cells are affected, the disease is called myeloid leukemia.
  • Chronic myeloid leukemia outbreaks as cells in the maturity period mutate.
  • AML Acute myeloid leukemia
  • AML is a kind of blood cancer characterized by the unlimited growth and suppression of differentiation of blasts in a specific stage of cell differentiation.
  • AML shows various genetic mutations which are known to be closely related to the response to anticancer therapy as well as to the prognosis thereof.
  • cytogenetic methods are widely used for analyzing chromosomal variations and classfying
  • AML AML patients with t(8;21) (translocation between the 8 th and 21 st chromosomes), t(15;17)
  • molecular analysis methods are also used to examine the relationship between gene mutation, or alteration of expression, and prognosis.
  • Patients having internal tandem duplication mutations of FLT3 (fms-like tyrosine kinase 3) or an increased expression level of EVl (ectotropic viral integration 1 site) gene are known to have a bad prognosis.
  • CEBPA CCAAT/enhancer binding protein alpha
  • Leukemia therapy is very complicated and depends on the type of leukemia. Clinically, leukemia patients who show resistance to therapy have very short survival time. Accordingly, it is very significant to form a proper treatment scheme from the various therapies, such as radiation therapy, operation therapy, chemical therapy, etc., and to apply it to patients in view of reducing expensive medical fees as well as mental and physical pain. Of the patients whose illnesses are diagnosed as the same cancer, some may respond positively to a drug whereas others may show no response to the same drug. In addition, the response to the same drug, even though positive, may differ in level from one patient to another. Thus, predictability of the response of patients to drugs endows the physician with the ability to forego unnecessary treatments, thereby increasing the efficacy and effectiveness of the treatment used.
  • therapies such as radiation therapy, operation therapy, chemical therapy, etc.
  • FIG. 1 is a graph showing p values, obtained in a log rank test for examining the significance of recurrence difference between a low risk group and a high risk group, which are divided according to the risk score of AML patients.
  • FIG. 2 is a Kaplan-Meier plot showing the recurrence frequency in the two groups of Fig. 1, wherein 33 of the total of 55 AML patients are in a low risk group.
  • the present invention pertains to a marker for use in predicting the response of AML patients to anticancer drugs .
  • the present invention pertains to a kit for predicting the response of AML patients to anticancer drugs, comprising an agent for measuring the expression of a marker gene capable of predicting the response of AML patients to anticancer drugs.
  • the selected marker genes are additionally found to be useful as predictors for the therapeutic possibility of anticancer drugs in AML patients in advance of the administration thereof.
  • the present invention is directed to a kit for predicting the response of AML patients to anticancer drugs, comprising an agent capable of analyzing the expression of an anticancer therapy predictive marker gene selected from among the marker genes .
  • ⁇ AML Acute Myeloid Leukemia
  • myloid line a malignant blood disease, characterized by the cancerous proliferation of the white blood precursor cells of the myloid line, which accumulate in organs and interfere with the production of normal blood cells when the balance in the bone marrow is broken and thus invade the peripheral blood or other organs.
  • ⁇ marker for predicting the response to anticancer drugs' means a material which is used to determine whether or not an anticancer drug is useful in the treatment of cancer in advance of the administration thereof by way of the measurement of the expression level on the basis of which the response to the anticancer drug is predictable.
  • the marker may include organic biomolecules such as nucleic acids, polypeptides, proteins, lipids, and saccharides.
  • the marker for predicting the response to anticancer drugs therapy is selected from among nucleic acids and polypeptides, which are predictive of the response of AML patients to anticancer drugs.
  • the expression level of the marker can be determined by quantitatively measuring the mRNA or protein thereof, with preference for mRNA.
  • techniques, useful in the present invention, for determining the expression level of mRNA include, but are not limited to, DNA chip, RT-PCR, competitive RT-PCR, real-time RT-PCR, RPA (RNase protection assay), and Northern blotting techniques.
  • a DNA chip in which a marker gene or fragments thereof are attached at high density on a substrate, such as glass, is used for measuring the expression level of mRNA.
  • the DNA chip requires a nucleotide, labeled with, for example, a fluorescent constituent at its terminus or an intermediate site, for hybridization.
  • the nucleotide may be cDNA synthesized from mRNA prepared ' from a sample of interest. Following the hybridization of cDNA on the DNA chip, the expression level can be readily read.
  • RT-PCR may be used to determine the expression level of mRNA.
  • RT-PCR is a technique in which mRNA, prepared from a sample of interest, is
  • PCR PCR
  • the expression of the marker gene can be determined.
  • the protein of the gene may be quantitatively analyzed with an antibody which specifically binds to the protein.
  • analytical techniques using antibodies include Western blotting, ELISA (Enzyme Linked Immunosorbent Assay) , RIA (Radioimmunoassay) , radioimmunodiffusion, rocket Immunoelectrophoresis, immunohistochemical stain, immunoprecipitation assay, complement fixation assay, FACS, protein chip, etc., but are not limited thereto.
  • the kit for use in measuring the expression of the marker gene may comprise an additional constitutional element suitable for the analysis technique used.
  • a DNA chip kit may comprise a substrate to which a gene of interest or the cDNA thereof is attached, a reagent for fluorescent labeling, and an enzyme.
  • An RT-PCR kit may comprise a pair of primers specific for the gene. Each of the primers is a nucleotide sequence, specific for the marker gene, having a length from 7 to 50 bp.
  • the RT-PCR kit may comprise a container, a reaction buffer, deoxyribonucleotide triphosphates (dNTPs) , Taq-polymerase, reverse transcriptase, DNAse, or an RNAse inhibitor.
  • dNTPs deoxyribonucleotide triphosphates
  • the kit according to the present invention can be applied to anticancer drugs for use in the treatment of AML.
  • naturally occurring materials and pure chemicals prepared therefrom as well as synthetic organic chemicals for general use in chemical therapy can be analyzed in advance for therapeutic response using the kit of the present invention.
  • the anticancer drugs can be administered in combination.
  • idarubicin may be administered in combination with 4N-bihenoyl-l-beta- D-arabino-furanosylcytosine (BHAC) , daunorubicin with BHAC, cytarabine with one selected from among doxorubicin, daunorubicin, mitoxanthrone and thioguanine, mercaptopurin with methotrexate, mitoxanthrone with etoposide, asparaginase with vincristine, daunorubicin and prednisone, cyclophosphamide with vincristine, cytarabine and prednisone, cyclophosphamide with vincristine and thioguanine, daunorubicin with cytarabine and thioguanine and daunorubicin with vincristine and prednisone.
  • BHAC 4N-bihenoyl-l-
  • the present invention provides a method for predicting the response of AML patients to anticancer drugs, comprising the steps of 1) determining the expression level of a marker gene for predicting the response to an anticancer drug in a bio- specimen of an AML patient, the marker gene being selected from a group consisting of GENX-3414, SPINK2, PECAMl, AQP5, LAPTM5, DDX48, F2RL3, SLC3A2, GPR51, ZC3HDC8.
  • WASFl PSCD4, CDKNlA, AIFl, ADFP, CD164, RAB8A, ID2, ITGB2, MGC3047, MYST3, MT3, MGATl, BC002942, EVI2B, CPVL, MXl, LGALSl, MX2, KRTl, MICA, GABBRl, TINF2, MPV17, RXRA, JARIDlD, C2orf22, DPT, NDUFS2, CCIN, MADH3, NAG, E2F5, SMC6L1, CASPl, LILRBl, TCF4, TNPO3, DNCIl, TIE, ADM, SMUGl, PPPlCA, SALL3, IRF2, KIAA1223, NCOA3, CKS2, ZNF226, CTBS, MetRS, MGC5395, FLJ10349, CD4, EHD4, ARL4A and DNAJClO, and 2) statistically analyzing the expression level of the gene to estimate risk scores R.
  • the bio-specimen useful for measuring the expression level of the marker gene for predicting the response to anticancer drugs in step 1) may be exemplified by bone marrow, lymph nodes, spleens, blood and lymph fluid, with preference for bone marrow.
  • the method may further comprise the step of conforming the risk score R with reference to a database in which risk scores R of AML patients have been accumulated.
  • the risk score R can be calculated using the HR (Hazard ratio) or ⁇ coefficient derived from Cox's proportional hazard modeling.
  • HR Hazard ratio
  • ⁇ HR' means the increment of the risk score R when a coefficient increases by 1, and corresponds to e p .
  • the risk score R of a patient can be calculated using the following mathematical formula 1, which expresses the sum of the individual products of the extent of expression of each gene and the coefficient ⁇ for each patient.
  • R stands for risk score
  • ⁇ for the beta coefficient of gene i
  • G ⁇ for the extent of expression of gene i.
  • a four genes selected in duplicate are represented licate
  • b Hazard ratio, the increasing hazard when gene expression is increased two-fold.
  • An HR of 2 means that the hazard level doubles when gene expression is increased two-fold.
  • c mean deviation of gene expression
  • d GenBank accession No.
  • e UniGene Cluster ID.
  • R risks scores
  • both the method for measuring the expression rate of a marker gene and the anticancer drugs described above can be used.
  • EXAMPLE 1 Assay for Gene Expression in the Bone Marrow Sample from AML Patients Using DNA Chip
  • RNA was isolated from bone marrow samples of 55 AML patients. For this, first, 1 ml of a bone marrow sample was mixed with 5 ml of a TriZol reagent (InVitrogen, Cat. No. 15596-018), followed by rupturing the cells with a tissue homogenizer. The subsequent RNA isolation procedure was conducted according to the protocol provided by the manufacturer of the TriZol reagent. The RNA thus obtained was further purified using an RNeasy kit (Qiagen, Cat. No. 74106) according to the protocol provided by the manufacturer thereof.
  • TriZol reagent InVitrogen, Cat. No. 15596-018
  • RNA was quantified by measuring absorbance at 260 nm using a spectrophotometer.
  • reference RNA was also used for hybridization on a DNA chip.
  • the reference RNA was isolated from cell lines based on blood cells. In detail, the seven cell lines HL-60, K-
  • RNA-CEM CCRF-CEM
  • CEM-CM3 CEM-CM3
  • Molt-4 THP-I
  • RNA fragments were mixed in equal amounts to give the reference RNA.
  • the DNA chip for use in the assay was a 16K human cDNA chip comprising 15,972 cDNA probes (Digitalgenomics, Korea).
  • the DNA chip was prepared as follows. First, a plasmid carrying the cDNA was isolated from a stock of bacteria, and probe sequences were amplified from the plasmid using PCR. The cDNA thus obtained was purified with a PCR Clean-Up kit and dissolved at a concentration from 100 to 200 ng/ ⁇ l in a spotting solution containing 50% DMSO. After the cDNA solution was spotted on GAPS II slides
  • RNA isolated from 10 ⁇ g of a marrow sample and the reference RNA were reverse transcribed into cDNA in the presence of aminoallyl-dUTP, and the cDNA was then labeled with Cy5 and Cy3 through reaction respectively with monoester-Cy5 and monoester-Cy3.
  • the labeled samples were purified using a PCR Clean-Up kit and subjected to hybridization for 16 hours or longer on the DNA chip. Then, the DNA chip was washed with a washing solution of SSC so as to remove non-specific hybrids. The washed DNA chip was scanned with a confocal laser scanner (Perkin Elmer, Scanarray Lite) to collect fluorescence data from each spot.
  • a confocal laser scanner Perkin Elmer, Scanarray Lite
  • EXAMPLE 2 Data about Gene Expression of AML Patient and Response to Anticancer Drugs
  • a F stands for female and M for male.
  • b 1 was marked for the patients whose conditions could not be monitored for reasons other than the disease or who did not show disease recurrence before the final monitoring time, and 0 for the patients who suffered from disease recurrence.
  • c The period from anticancer therapy to recurrence is indicated as months, d: subtype of AML according to French-American-British classification.
  • a bone marrow samples taken from the AML55 patients were measured for the expression level of each of the marker genes for predicting the response to anticancer drugs.
  • the risk score R of the AML 55 patients was calculated using the hazard ratio or ⁇ coefficient obtained from the Cox' s proportional hazard model .
  • the term "hazard ratio" has the same value as e p and refers to the increase proportion of risk when a variable is increased by 1.
  • the hazard score R of a patient was calculated according to the following mathematic formula 1,
  • the risk score R of a patient can be calculated using the following mathematical formula 1, which expresses the sum of the individual products of the extent of expression of each gene and the coefficient ⁇ .
  • Calculated risk scores of the AML 55 patient are given in Table 3.
  • the risk score of the AML 55 patients is found to be -23.29, as calculated using 67 marker genes for predicting response to anticancer drugs. From the expression levels of the 67 marker genes, obtained with respect to the AML55 patients, the risk scores were calculated, and the results are listed in Table 3.
  • EXAMPLE 3 Assay for Prediction of Response to Anticancer Drugs Using the Expression of Marker Gene
  • Leave-one-out cross-validation was conducted to predict the response of AML patients to the anticancer drugs using the expression of the marker genes selected according to the method described in Example 2, and to evaluate the prediction.
  • Leave-one-out cross-validation is a statistical technique in which after the data of one patient is excluded from the total data set and marker genes are selected, the response of the patient to anticancer drugs can be predicted using the expression of the marker genes in the patient. The repetition of this procedure as many times as the total number of patients allows an independent prediction to be made for each individual patient. Using the risk scores obtained from the independent prediction, survival curves are compared between a high risk patient group and a low risk patient group such that the significance of prediction for the response of individual patients to anticancer drugs can be determined.
  • the patients can be classified into a high risk group and a low risk group.
  • a Kaplan-Meier plot which provides recurrence curves of the two groups, was generated while the number of the members in the low risk group was increased by one in order of increasing risk.
  • a log rank test was performed to examine whether there was a significant difference between the recurrence curves of the two groups .
  • FIG. 1 is a graph showing p values obtained in the log rank test.
  • the Kaplan-Meier plot generated at this time is shown in FIG. 2.
  • Marker genes useful in predicting the response of AML patients to anticancer drugs can be selected in accordance with the present invention. Accordingly, when biological samples taken from AML patients are applied to a kit comprising an agent for measuring the expression of the marker genes for predicting the response of AML patients to anticancer drugs, the expression patterns of the genes can be analyzed so as to predict the response of the AML patients to anticancer drugs .
  • the present invention can give information on suitable chemotherapy for AML patients, thereby improving the therapeutic effect of the anticancer drugs used and relieving the pain and economic burden of the patients.

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Abstract

L'invention concerne un marqueur apte à prédire la réponse de patients souffrant de leucémie aiguë myéloïde (LAM) à une thérapie anticancéreuse. La présente invention porte également sur un kit comprenant un moyen pour mesurer le niveau d'expression de gènes marqueurs aptes à prédire cette réponse, afin de prédire la réponse de patients LAM à des médicaments anticancéreux. L'invention concerne en outre une méthode pour prédire la réponse de patients LAM à des médicaments anticancéreux sur la base de l'étendue de l'expression des gènes marqueurs. L'application d'un échantillon biologique extrait d'un patient LAM au kit pour analyser les modèles d'expression des gènes permet de prédire la réponse de patients LAM à des médicaments anticancéreux. Cette méthode peut donner des informations sur une chimiothérapie appropriée pour les patients LAM, améliorant ainsi l'effet thérapeutique des médicaments anticancéreux utilisés et allégeant la souffrance et le poids économique supportés par les patients.
PCT/KR2006/003822 2005-09-27 2006-09-26 Marqueurs pour predire la reponse d'un patient souffrant de leucemie aigue myeloide a des medicaments anticancereux Ceased WO2007037611A1 (fr)

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CN200680044375.1A CN101326290B (zh) 2005-09-27 2006-09-26 用于预测急性髓系白血病病人对抗癌药物应答的标记物
JP2008533238A JP5161091B2 (ja) 2005-09-27 2006-09-26 急性骨髄性白血病患者の抗癌剤治療反応性予測用マーカー

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KR10-2005-0089785 2005-09-27
KR1020050089785A KR100617467B1 (ko) 2005-09-27 2005-09-27 급성 골수성 백혈병 환자의 항암제 치료 반응성 예측용마커

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CN109321658A (zh) * 2018-11-23 2019-02-12 浙江大学 一种检测宫颈癌易感性的试剂盒
EP3411517A4 (fr) * 2016-02-06 2020-01-01 University Health Network Procédé d'identification de patients à haut risque souffrant de lam
WO2021118243A1 (fr) * 2019-12-10 2021-06-17 아주대학교산학협력단 Composition faisant intervenir un auto-anticorps wasf2 pour le diagnostic précoce du carcinome hépatocellulaire
US11111542B2 (en) 2016-02-06 2021-09-07 University Health Network Method for identifying high-risk AML patients
CN114317532A (zh) * 2021-12-31 2022-04-12 广东省人民医院 用于预测白血病预后的评估基因集、试剂盒、系统及应用
CN114540490A (zh) * 2021-06-24 2022-05-27 山西高等创新研究院 Lcdr作为预防和/或治疗癌症的药物的治疗靶点中的应用

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US20100311606A1 (en) * 2007-05-07 2010-12-09 Liewei Wang Pharmacogenomic cell line panel and use thereof
WO2011085653A1 (fr) * 2010-01-12 2011-07-21 中国科学院上海生命科学研究院 Procédé et trousse d'identification et de traitement de la maladie de graves
EP2589665A4 (fr) * 2010-06-29 2013-11-20 Univ Kurume Procédé de prédiction de l'effet thérapeutique d'une immunothérapie sur un patient cancéreux, et ensemble de gènes et kit à utiliser dans le procédé
CN102146441B (zh) * 2010-12-30 2013-07-10 苏州大学 一种检测急性髓系白血病易感性的试剂盒
CN104931704B (zh) * 2014-03-19 2016-11-09 中国人民解放军军事医学科学院放射与辐射医学研究所 脂肪分化相关蛋白作为hbv-hcc患者筛查标记物的新用途
EP4038205B1 (fr) * 2019-10-02 2024-07-03 Ophiomics - Investigação e Desenvolvimento em Biotecnologia, SA Marqueurs de pronostic clinique et moléculaire pour une transplantation hépatique
CN111596067B (zh) * 2020-06-03 2021-06-22 四川大学华西第二医院 Zc3h8在pop早期预警、诊断、预后评估中的应用和产品

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