[go: up one dir, main page]

WO2004065632A1 - Procede de diagnostic de tumeurs et de metastases - Google Patents

Procede de diagnostic de tumeurs et de metastases Download PDF

Info

Publication number
WO2004065632A1
WO2004065632A1 PCT/JP2004/000386 JP2004000386W WO2004065632A1 WO 2004065632 A1 WO2004065632 A1 WO 2004065632A1 JP 2004000386 W JP2004000386 W JP 2004000386W WO 2004065632 A1 WO2004065632 A1 WO 2004065632A1
Authority
WO
WIPO (PCT)
Prior art keywords
cancer
metastasis
tumor
nucleic acid
expression
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Ceased
Application number
PCT/JP2004/000386
Other languages
English (en)
Japanese (ja)
Inventor
Akira Takeuchi
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Individual
Original Assignee
Individual
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Individual filed Critical Individual
Publication of WO2004065632A1 publication Critical patent/WO2004065632A1/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

Links

Classifications

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

Definitions

  • the present invention relates to a method for determining or evaluating a tumor or a cancer by observing an increase or decrease in the expression level of an angiogenic factor in blood cells such as monocyte cells and Mactophage.
  • Cancer mortality is extremely high in men and women, and although much attention has been paid to cancer diseases, mortality has not declined significantly in recent years. Early detection of cancer is extremely important in determining survival and will be an important issue in the future. Cancer cells sometimes produce substances that are undetectable or found only at very low levels in healthy individuals. This feature could be used to develop innovative diagnostic techniques. Using these techniques, testing methods for substances produced by cancer cells have been developed. Tests aimed at detecting cancer-binding substances are also expected to be useful in discriminating between individuals with cancer in comparison to healthy individuals. As a representative example, one type of marker for metastatic staging of cancer is the presence of an oncogene 'allele.
  • genes associated with more than 30% of breast and ovarian cancers have been identified as neu / HER2 / c-erbB2 proto-oncogenes.
  • the extent of the proto-oncogene and the degree of overexpression of its protein product may be attributed to the severity of the disease. They have been shown to correlate with prognostic signs (S'lamon et al., Science 244: 707-712 (1989)).
  • abnormal alleles of the ras and myc gene families have been shown to be involved in human cancer progression, and some researchers speculate that they are useful as prognostic indicators ( Field. JK et al., Anticancer Res. 10: 1-22 (1990)).
  • Knowing the metastatic potential of a tumor is important in developing cancer treatments that maximize patient survival and quality of life.
  • the most aggressive treatment regimen is rapid tumor growth It should be reserved for patients at very high risk of multiplication and metastasis.
  • angiogenesis is essential for cancer progression.
  • Angiogenesis is the development of new capillaries from pre-existing blood vessels. Normally, mammalian angiogenesis is limited to the reproductive system, embryogenesis, and repair following injury. However, angiogenesis can also occur in pathological conditions such as cancer, retinal neovascularization, neovascularization in atheroma plaques, hemangiomas, arthritis and psoriasis.
  • tumors can remain in place as small asymptomatic lesions for many years. Perfusion provides a large supply of oxygen and nutrients to tumors that lead to angiogenesis. Thus, angiogenic tumors can grow and proliferate. Tumors must constantly stimulate the growth of new capillaries to keep growing. In addition, angiogenesis causes tumor cells to enter the host animal's circulatory system. New blood vessels provide a way for tumor cells to enter the circulation and metastasize to distant sites.
  • the extent of neovascularization is strongly correlated with metastasis in primary breast, bladder, prostate, non-small cell lung, cutaneous melanoma, and cervical cancer.
  • tumor specimens were analyzed histologically.
  • the extent of tumor mass angiogenesis is an independent predictor of metastatic potential and has been shown to be more reliable than other prognostic markers, but its number is easy to measure given any cancer or tumor Not something.
  • the value of markers for this metastasis also varies greatly depending on the type of tumor, making it difficult to determine at present. That is why many people die from reoccurrence due to metastasis It is the current situation.
  • cancer or tumor can be determined from the expression levels of nucleic acids by the beta inhibitor, collagenase, ⁇ (matrix meta-oral protease) and ⁇ -kinase factor genes.
  • bFGF basic fibroblast growth factor
  • VEGF vascular endothelial growth factor
  • CD147 / EMMPRIN insulin growth factor 2
  • CD147 / EMMPRIN insulin growth factor 2
  • colony stimulating factor a factor that is candidates for angiogenesis metastasis markers.
  • Other factors that are candidates for angiogenesis metastasis markers include oral kinase-type plasminogen activator and plasminogen activator inhibitor 1, as well as various collagenases and Perkinase n
  • the factor or factors that cause tumor angiogenesis have not yet been determined, but bFGF and VEGF are presumed to be synergistic.
  • bFGF levels were elevated in urine in approximately 37% of a wide range of cancer patients. Similarly, bFGF was elevated in the serum of 10% of such patients. The highest levels of bFGF were observed in patients with metastatic disease. bFGF is abnormally high in cerebrospinal fluid in children with brain tumors, and these high levels correlate with microvessel density in tumor specimens.
  • Angiogenesis in cancer tissue is difficult to determine because of the complex relationship between a wide variety of neogenesis factors and other components. In order to perform the procedure on cancerous tissue, it is necessary to remove the cancerous tissue, and the method is limited. It is therefore an object of the present invention to provide an objective judgment tool for characterizing and staging the progression of a disease state using the specific properties of a cancer or tumor.
  • the present invention takes advantage of the abnormal growth of cells due to cancer or tumors that triggers initiation of angiogenesis and the like.
  • the condition of the cancer is detected and determined by examining the expression of a nucleic acid by a specific gene from blood cells of the subject.
  • the information obtained by this diagnostic tool should be used by physicians to plan treatment protocols to treat and manage these disease states.
  • This expression indicates that the level of VEGF could be a molecular marker for the progression of the cancer state, and we have now found that this expression of VEGF also occurs in monocytes.
  • the expression of receptor proteins on endothelial cells associated with tumors that respond to VEGF and other factors can also be detected and used to diagnose.
  • receptor proteins include, but are not limited to, KDR / flk-1, fit-1, CD147 / EMMPRIN and / or tek / tie-2. Co-expression of VEGF and any of these receptor proteins is also indicative of a cancer or tumor where growth and metastasis may be increased. Identification of specific receptors involved in the angiogenesis pathway is important in determining which treatments are most effective and selective at inhibiting angiogenesis.
  • VEGF is a heparin-binding homodimeric glycoprotein of about 45 kD. VEGF has been isolated from cell lines of various origins and its genes have been cloned from cells of other species as well as human cells. VEGF gene transcripts are spliced into any of at least four different molecular species consisting of 121, 165, 189, and 206 amino acids. VEGF 165 is the most common form.
  • VEGF receptor molecules KDR / flk-1 and / or fit-1 KDR / flk-1 and flt-1 are both receptor tyrosine kinase proteins with high specificity for VEGF. Those ligands bind These receptors then dimerize and transmit intracellular signals by tyrosine phosphorylation.
  • VEGF vascular endothelial growth factor
  • endothelial cells proliferate rapidly by dividing at most every 5 days.
  • Other receptors observed during angiogenesis are fll-4 receptors, fibroblast growth factor receptors, platelet-derived growth factor receptors, and epithelium. Growth factor receptor, and the Met oncogene.
  • the present invention can evaluate not only the cancer or tumor but also the metastatic stage of the cancer, and can examine the course of treatment in a method for devising an appropriate treatment. If it is an indicator of improvement, it is very effective in preventing recurrence due to metastasis of cancer that seems to have been cured once.
  • the key to the method of the present invention lies in the discovery that expression of genes related to cancer or tumors is caused by blood cells.
  • a sample of blood cells is molecularly characterized for the presence of VEGF messenger RNA or protein using the techniques described below. Using a similar technique, the sample is also characterized for expression of factors HIF-1, KDR / flk-1, fit-1 and / or tek / tie-2.
  • FIG. 1 shows the expression of VEGF and CD147 / EMMPRIN by ⁇ -PCR amplification expressed on monocytes.
  • FIG. 2 shows the expression levels of VEGF, CD147 / EMMPRIN, and MMP by PT-PCR amplification expressed in monocytes, with the intensity of GAPDH being 100.
  • Fig. 3 shows the expression levels of various factors by the expressed PT-PCR. Best mode for carrying out the invention
  • the expression of any of a number of oncogenes known to be involved in cancer, including SAM, SIS, SRC, and TPK, can also be analyzed.
  • tumors such as RB1, p53, WT1, DCC, NFL1, FAP, and MEN-1
  • the expression of the suppressor gene can also be assessed.
  • the level of gene expression can be determined by using nucleic acid from the cell as a starting point for such an assay technique, which can be isolated according to standard nucleic acid preparation procedures well known to those skilled in the art.
  • RNA of blood cells suspected of expressing the VEGF gene can be isolated and tested using hybridization or PCR techniques as described below.
  • Isolated cells can be derived from cell culture or from patients, but generally can be easily separated from blood. Granulocytes, leukocytes, and macrophages can also be easily separated from conventional methods.
  • One or more labeled nucleic acid reagents including recombinant DNA molecules or in vitro transcribed antisense RNA probes, from RNA obtained from a sample or nucleic acid such as cDNA generated from RNA, and these reagents Contacting and incubating under conditions suitable for specifically annealing to the complementary sequence within the gene can be included.
  • these nucleic acid reagents are at least 15 to 30 nucleotides in length. After incubating, remove any unannealed nucleic acids from the nucleic acid: VEGF molecule hybrid. The presence of the hybridized nucleic acid is then detected if such a molecule is present.
  • Diagnostic methods using patient blood cells or monocytes, granulocytes, leukocytes, and macrophages include, for example, PCR (Mullis, KB, 1987, an experimental embodiment described in US Pat. No. 4,683,202) after amplification of those molecules. Detecting the amplified molecule using techniques well known to those skilled in the art. To determine whether the expression of the nucleic acid by the gene was up-regulated, the resulting wide sequence was ⁇ ⁇ ⁇ Can be compared to what would be expected if the broadened nucleic acid contained only normal levels of gene transcript.
  • cDNA is synthesized from the RNA of interest (eg, by reverse transcribing an RNA molecule to cDNA).
  • a nucleic acid amplification reaction such as a PCR amplification reaction is performed using the sequence in the cDNA as a type III.
  • the nucleic acid reagent used as a synthesis initiation reagent is selected from the nucleic acid sequences of the respective factors.
  • the preferred length of such a nucleic acid reagent is at least 9 to 30 nucleotides.
  • nucleic acid amplification can be performed using radioactively or non-radioactively labeled nucleotides.
  • sufficient amplification product can be produced that can be visualized using standard bromide staining or other suitable nucleic acid staining methods.
  • Reverse transcriptase reactions and PCR amplification were performed in a Perkin Elmer 960 PCR machine (Erneryville, CA) without interruption. 20 // 1 0 £? (Jechirupi mouth carbonate) 40 011 ⁇ 1 1 in treated water? Immediately before adding the R reagent, it was placed in a water bath at 65 ° C for 5 minutes, and then quickly cooled on ice.
  • the total PCR volume of 50 ⁇ l is 2.5 units of Taq Holimerase, Perkin Elmer, Emeryvi I 1e, CA), and 2 units of AMV reverse transcriptase (Boehringer Mannheim, I ndianapolis, IN), dCTP, dATP, dGTP and dTTP (Perkin Elmer, Emerryvi 1) le, CA), 18 pM of each primer, 10 mM Tris-HCI) 50 mM KCI) 2 mM MgC12 (Perkin Elmer) Emeryvi 11 e, CA).
  • the PCR conditions were as follows.
  • Cycle 1 is for 15 minutes at 42 ° C, then 197 ° C for 15 seconds (1 cycle):
  • Cycle 2 is for 1 minute at 9.5 ° C, then 1 minute at 60 ° C. 2 ° C for 30 seconds (15 cycles):
  • Cycle 3 is 95 ° C for 1 minute, followed by 60 ° C for 1 minute and 72 ° C for 1 minute (10 cycles):
  • Cycle 4 is 9 5 ° C for 1 minute, followed by 60 ° C for 1 minute and 72 ° C for 2 minutes (8 cycles):
  • Cycle 5 is 72 ° C for 15 minutes (1 cycle): The final cycle is when the sample is mechanical. It was stored at 4 ° C until it was removed from the container.
  • the PCR product of 501 was concentrated to 10 ⁇ l by vacuum centrifugation, and the whole sample was electrophoresed in a thin 3% trisodium borate-EDTA containing ethyl bromide ( ⁇ ⁇ ⁇ ). All specimens were analyzed at least twice to confirm positive or negative results.
  • the risk of possible false positives arising from cross-contamination was determined by performing RT- ⁇ CR in a single tube without interruption and using a filtered tip.
  • the use of large amounts of Taq polymerase greatly extended the elongation time, and enhanced sensitivity by analyzing all 501 PCR products on a thin agarose gel containing ethidium bromide.
  • Figures 1 and 2 show the expression of nucleic acids in blood cells of cancer patients.
  • Patient 1 Before treatment for cancer patients, Patient 2 Treatment for cancer patients 3 months, Patients Treatment for cancer patients 6 months, N healthy individuals, P cancer patients 2.
  • Higher levels of VEGF and CD147 / EMMPRIN expression were found in monocytes of cancer patients compared to healthy N (Patient 1, P). It was also found that its expression gradually weakened during the treatment of cancer (Patient 2, 3).
  • VEGF in plasma was in the normal range of 25.3 pg / ml before treatment (less than 38.3 Pg / ml), and after treatment, it was 34.4 pg / ml, indicating that the condition of cancer could not be determined. Did not.
  • VEGF expression in the blood cells decreased with each treatment, and it was in fact healing.
  • Examining gene expression can be a convenient means of determining cancer or metastasis of cancer that was not revealed by plasma serum.
  • blood cells are fractionated into monocytes, granulocytes, leukocytes, and macrophages by a conventional method, and bFGF, VEGF, CD147 / EMMPRIN, KDR / flk-1, fit-1, tek / tie-2, FC y receptor, insulin growth factor (IGF), platelet-derived growth factor, colony stimulating factor, perokinase-type plasminogen activator, plasmaminogen activator inhibitor, collagenase, MMP (matrix The expression of nucleic acids by the respective genes in healthy subjects and cancer patients was examined for T.
  • IGF insulin growth factor
  • platelet-derived growth factor colony stimulating factor
  • perokinase-type plasminogen activator perokinase-type plasminogen activator
  • a physician can design an appropriate treatment tailored to the molecular stage of the patient's cancer state.
  • the present invention Using the method to measure the molecular profile of a tumor allows physicians to plan appropriate treatments to optimize both cancer treatment efficacy and patient quality of life. If the blood cells isolated from the patient are found to have increased VEGF expression using the diagnostic methods of the invention, the patient is a candidate for treatment for cancer or tumor. This leads to earlier detection of cancer or tumor, and early detection of cancer or tumor due to metastasis that once recurs. It is expected that early detection will save more people.

Landscapes

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

Abstract

L'invention porte sur une méthode pratique d'évaluation de cancers et de métastases par examen de la quantité d'un acide nucléique exprimée par un globule sanguin, et sur une méthode de diagnostic de cancers par examen de l'expression d'un acide nucléique. Il est difficile de diagnostiquer un cancer par les procédés usuels qui requièrent l'essai d'une protéine dans un sérum. Il existe par contre une méthode pratique de diagnostic des cancers et de leurs métastases consistant à mesurer l'expression d'un acide nucléique dans des monocytes ou des macrophages, globules sanguins présents dans le sérum, sans devoir rechercher la tumeur elle-même par biopsie ou d'autres moyens. Cette méthode de diagnostic se base sur la découverte que les tumeurs et leurs métastases agissent sur les cellules sanguines, en particulier sur les monocytes et les macrophages, où ils provoquent l'expression d'un acide nucléique dont l'examen rend le diagnostic aisé.
PCT/JP2004/000386 2003-01-21 2004-01-19 Procede de diagnostic de tumeurs et de metastases Ceased WO2004065632A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
JP2003-48222 2003-01-21
JP2003048222 2003-01-21

Publications (1)

Publication Number Publication Date
WO2004065632A1 true WO2004065632A1 (fr) 2004-08-05

Family

ID=32767750

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/JP2004/000386 Ceased WO2004065632A1 (fr) 2003-01-21 2004-01-19 Procede de diagnostic de tumeurs et de metastases

Country Status (1)

Country Link
WO (1) WO2004065632A1 (fr)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2013110817A1 (fr) * 2012-01-27 2013-08-01 Vib Vzw Biomarqueurs monocytaires utilisables en vue de la détection du cancer

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
MATSUNAGA H. ET AL: "Application of differential display to identify genes for lung cancer detection in peripheral blood", INT. J. CANCER, vol. 10, no. 5, 2002, pages 592 - 599, XP002979352 *
QI Z.L. ET AL: "Expression and clinical significance of surfactant protein D mRNA in peripheral blood of lung cancer patients", CHINESE JOURNAL OF CANCER, vol. 21, no. 7, 2002, pages 772 - 775, XP002979353 *

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2013110817A1 (fr) * 2012-01-27 2013-08-01 Vib Vzw Biomarqueurs monocytaires utilisables en vue de la détection du cancer
AU2013213563B2 (en) * 2012-01-27 2018-04-19 Katholieke Universiteit Leuven, K.U.Leuven R&D Monocyte biomarkers for cancer detection
US10041126B2 (en) 2012-01-27 2018-08-07 Vib Vzw Monocyte biomarkers for cancer detection

Similar Documents

Publication Publication Date Title
US20090029372A1 (en) Adam12 as a biomarker for bladder cancer
JP2011515109A (ja) ＦＩＳＨ法を用いた循環腫瘍細胞中のＩＧＦ１Ｒ／Ｃｈｒ１５を検出するための方法
CN105543408B (zh) 冠心病早期诊断标志物
JP2012085556A (ja) 乳がんの診断方法
JP5209272B2 (ja) 肝臓癌関連遺伝子、及び肝臓癌リスクの判定方法
KR20120132592A (ko) 실시간 중합반응을 이용한 암의 진단을 위한 정보제공방법 및 이를 위한 암 진단용 키트
JP7187081B2 (ja) 液体生検多重癌遺伝子バイオマーカーを用いた乳癌の早期診断および治療後のモニタリング方法
JP6638128B2 (ja) 腎がんの悪性度の検査マーカー及び検査方法
CN113631723B (zh) 结核病标志物在结核病诊断和疗效评估中的应用
CN106967810A (zh) 一种检测fgfr3基因突变以诊断膀胱癌的方法及试剂盒
US20230212692A1 (en) Method for sorting colorectal cancer and advanced adenoma and use of the same
JP2012085555A (ja) 乳がん診断用マーカー
CA2872245A1 (fr) Test sanguin de depistage du cancer utilisant de l'arn bc200 isole a partir de sang peripherique et utilisable en vue du diagnostic et du traitement du cancer du sein invasif
JP7610265B2 (ja) 大腸癌診断用マーカー、大腸癌の診断を補助する方法、大腸癌の診断のためにデータを収集する方法、大腸癌の診断用キット、大腸癌治療薬、大腸癌の治療方法、大腸癌の診断方法
WO2004065632A1 (fr) Procede de diagnostic de tumeurs et de metastases
JP2024035040A (ja) 分析方法、キット及び検出用デバイス
JP7299765B2 (ja) マイクロｒｎａ測定方法およびキット
US20160025728A1 (en) Brain-specific gene signature of tumor cells
KR20070116567A (ko) 위 암종으로부터의 림프절 전이의 검출법
WO2021060311A1 (fr) Procédé de détection d'une tumeur cérébrale
CN105861735A (zh) Rap1b在冠心病诊断中的应用
KR102548873B1 (ko) 대장암 및 진행 선종의 선별 검사 방법 및 그 응용
KR102540668B1 (ko) 양성 종양 또는 결절로부터 암을 구분하기 위한 바이오마커
JP2004151003A (ja) Ｍｕｃ１およびケラチン１９の検査方法
JP6017448B2 (ja) 血液疾患の診断方法

Legal Events

Date Code Title Description
AK Designated states

Kind code of ref document: A1

Designated state(s): AE AG AL AM AT AU AZ BA BB BG BR BW BY BZ CA CH CN CO CR CU CZ DE DK DM DZ EC EE EG ES FI GB GD GE GH GM HR HU ID IL IN IS JP KE KG KP KR KZ LC LK LR LS LT LU LV MA MD MG MK MN MW MX MZ NA NI NO NZ OM PG PH PL PT RO RU SC SD SE SG SK SL SY TJ TM TN TR TT TZ UA UG US UZ VC VN YU ZA ZM ZW

AL Designated countries for regional patents

Kind code of ref document: A1

Designated state(s): BW GH GM KE LS MW MZ SD SL SZ TZ UG ZM ZW AM AZ BY KG KZ MD RU TJ TM AT BE BG CH CY CZ DE DK EE ES FI FR GB GR HU IE IT LU MC NL PT RO SE SI SK TR BF BJ CF CG CI CM GA GN GQ GW ML MR NE SN TD TG

121 Ep: the epo has been informed by wipo that ep was designated in this application
122 Ep: pct application non-entry in european phase
NENP Non-entry into the national phase

Ref country code: JP

WWW Wipo information: withdrawn in national office

Country of ref document: JP