WO2007107334A1 - Procédé de diagnostic du cancer ex vivo - Google Patents

Procédé de diagnostic du cancer ex vivo Download PDF

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Publication number
WO2007107334A1
WO2007107334A1 PCT/EP2007/002461 EP2007002461W WO2007107334A1 WO 2007107334 A1 WO2007107334 A1 WO 2007107334A1 EP 2007002461 W EP2007002461 W EP 2007002461W WO 2007107334 A1 WO2007107334 A1 WO 2007107334A1
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WO
WIPO (PCT)
Prior art keywords
sample
compound
individual
fraction
cancer
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PCT/EP2007/002461
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English (en)
Inventor
Wassyl Nowicky
Askold Nowicky
Original Assignee
Nowicky Wassili
Askold Nowicky
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 Nowicky Wassili, Askold Nowicky filed Critical Nowicky Wassili
Publication of WO2007107334A1 publication Critical patent/WO2007107334A1/fr

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Classifications

    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57484—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumor, cancer, neoplasia, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides, metabolites
    • G01N33/57488—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumor, cancer, neoplasia, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides, metabolites involving compounds identifable in body fluids

Definitions

  • the present invention is in the field of medical diagnostics and relates to an ex vivo method of detection of a pathological condition, particularly of a malignant tumor, in a human or non-human mammalian individual.
  • blood cells particularly white blood cells
  • the cells of diseased patients differ from the ones of healthy donors inter alia in their ability to take up nutritional compounds such as, e.g. amino acids, from their natural biological environment as well as from an artificial environment such as a suitable ex vivo nutrient medium.
  • a fully ex vivo diagnostic method for the determination of a pathological condition in an individual was established using the different cellular characteristics of body liquids, e.g. of red and/or white blood cells of diseased patients, as compared to corresponding body liquids, e.g. to corresponding red and/or white blood cells of healthy donors, as a measure for the prediction of the presence of a malignancy, particularly of a malignant tumorigenic activity and most particularly for the detection of a cancer disease in an afflicted individual.
  • This ex vivo method is extremely patient-friendly as it does not require administration of any possibly hazardous agent to a patient solely for the purpose of cancer diagnostics.
  • amino acid as used herein relates to any known L- or D-amino acid known to occur in human or animal individuals, including all essential and non-essential amino acids and all other naturally occurring amino acids.
  • amino acid related compound mainly relates to modified amino acids, amino acid derivatives and metabolites, and amino acid-based compounds such as smaller peptides naturally occurring in the animal or human body.
  • compound fraction as used herein relates to an amino acid fraction, a fraction comprising an amino acid related compound or any other compound.
  • the present invention therefore in its first aspect relates to a method of detecting a pathological condition, particularly the presence of a malignancy, more particularly of a malignant tumor or a cancer disease, in an individual, the method comprising: - obtaining a sample of a bodily fluid from said individual;
  • the invention relates to such a method, wherein said body liquid sample is selected from the group consisting of a whole blood sample, typically a heparinised whole blood sample, a saliva sample, and a sweat sample.
  • the invention relates to such a method, wherein said method comprises separating said cell-free liquid fraction into single compound fractions by liquid chromatography, preferably by HPLC, and detecting at least one, preferably a plurality of compound fractions, present in said cell-free fraction, using a suitable detection system.
  • the invention relates to such a method, which comprises detecting in the control sample at least one compound fraction which is not detected, or only at a significantly lower concentration, in the sample obtained from a said individual.
  • This significantly lower concentration is typically in a range of equal or less than 20 %, preferably equal or less than 10%, of the corresponding values of a said compound fraction in a pooled control.
  • the invention relates to such a method, wherein under the analytical conditions referred to in Example 1 at least one of said compound fractions present in the control sample but not or at a significantly lower concentration in the sample of a said individual elutes at a retention time prior to aspartic acid.
  • the invention relates to such a method, wherein under the analytical conditions referred to in Example 1 one of said compound fractions present in the control sample but not or at a significantly lower concentration in the sample of said individual elutes at a retention time of about 2.5 min, e.g. at 2.57 min, and/or another one at about 3 min, e.g. at 3.07 min, and/or another one at about 3.7 min, e.g. at 3.79 min, while apartic acid elutes at about 4.3 min, e.g. at 4.34 min.
  • the invention relates to such a method which is suitable for early prognosis on the risk of cancer development in a said individual.
  • the invention relates to such a method, wherein the detection of a compound fraction present in the sample of a said individual at a concentration significantly lower than in a pooled control is indicative of an increased risk of developing cancer or of the presence of an early stage cancer in said individual.
  • the invention relates to such a method, wherein a failure to detect in the sample of a said individual a compound fraction present in a pooled control is indicative of the presence of a malignant tumor or of cancer cells or tissues in said individual.
  • the invention relates to such a method, wherein said compound fraction comprises a compound that elutes prior to aspartic acid under the analytical conditions referred to in Example 1 .
  • the invention relates to such a method which comprises detecting at least one compound fraction present in the experimental sample and not in a pooled control sample.
  • the invention relates to such a method, wherein under the analytical conditions referred to in Example 1 at least one of said compound fractions present in the experimental sample and not in the control sample elutes at a retention time later than that of alanine.
  • the invention relates to such a method, wherein under the analytical conditions referred to in Example 1 one compound fraction present in the experimental sample and not in the control sample elutes at a retention time of about 50 min and/or another one compound fraction present in the experimental sample and not in the control sample elutes at a retention time of about 70 min.
  • the present invention relates to the above described method which further comprises quantitative evaluation of the concentration of the amino acid or amino acid related compound corresponding to said product peak appearing ahead of the aspartic acid peak, for early prognosis on the risk of cancer development in said individual. If preliminary results hold true, it can be inferred from this method that if in a said individual a concentration level of said amino acid or amino acid related compound is detected of less than 20 %, particularly of less than 10 %, of the concentration level of a pooled control such low concentration is indicative of an increased risk of developing cancer or of the presence of an early stage cancer in said individual.
  • a detailed legend for the concentration profiles A and B is found in Table 1 and Table 2, respectively.
  • Fig. 6 represents an overlay of the profiles of Fig. 4 and Fig. 5;
  • Fig. 7 represents an enlarged and stretched view of a part of Fig.6.
  • a comparison of the chromatogram of the stomach cancer patient with the one of the healthy control represents that the peaks occurring at 5.35 min (peak 4), 10.35 min (peak 1 1 ), 23.23 min (peak 19; arginine), 33.99 min (peak 21 ; ⁇ -alanine) and 38.58 min (peak 24) are very low or entirely missing in the plot of the control while they are rather prominent in the plot of the cancer patient.
  • Example 1 Determination of metabolite profiles in blood samples of cancer patients and healthy donors
  • 5ml venous blood were taken from a lymphoma or prostate cancer patient and transferred to a heparinized glass vial. 1 ml thereof was transferred into an Eppendorf microcentrifuge glass container and centrifuged for 1 5 min at 1 500 g.
  • the such obtained plasma was deproteinized by admixing an equal volume of 1 M perchloric acid (HCIO 4 ) and centrifuging the mixture for 20 minutes at 1 2000 g.
  • HCIO 4 perchloric acid
  • As a control pooled venous blood from healthy donors was used and was subjected to the same procedure described above .
  • homotaurine Sigma Aldrich was used.
  • the protein- free extracts obtained by this procedure were stored at -20 0 C and kept for subsequent analysis.
  • Table 5 Presence/absence of low molecular weight compounds in blood plasma of lymphoma and prostate cancer patients
  • concentration values of some low molecular weight compounds, more specifically of some amino acids or amino acid related compounds determined from the plasma fraction of said blood samples exhibit significant differences in samples from healthy probands as compared to those from diseased patients, e.g. cancer patients. More specifically, whereas a product peak appears at a retention time of 2.57 min in the pooled plasma samples of the control group of healthy volunteers and not in those of the experimental group, two other product peaks appear at retention times of 57 and 70 min in the experimental samples (Table 5).
  • the peaks may not be attributed to any of the twenty natural amino acids usually present in a human body, but instead are assumed to be amino acid derivatives or related amino compounds, e.g. polar metabolic reaction products bearing at least one amino group.
  • the concentration profile i.e. the absence or presence of compounds at certain retention times under the given analytical conditions, obtained from the experimental samples as compared to the concentration profile obtained from the controls gives statistically relevant information as to the presence of malignancy in an individual.
  • Example 2 Determination of concentration profiles of selected amino acids and amino acid related compounds in blood samples of cancer patients and healthy donors Blood samples of a colon cancer patient and of a healthy donor have been prepared according to Example 1 . The plasma fractions obtained therefrom have been analysed according to Example 1 .
  • Table 6 Concentration profiles of selected amino acids and amino acid related compounds in blood plasma of a colon cancer patient and a healthy donor.
  • the concentration values of selected amino acids and amino acid related compounds determined from the plasma fraction of said blood samples exhibit significant differences in the control samples of the healthy proband as compared to the experimental samples of the diseased patient, i.e. cancer patient. Whereas some of the determined compounds have been found to have reduced levels in the experimental plasma sample as compared to the sample of the healthy volunteer, other compounds have been found to have increased levels, as can be seen in Table 6 and Figures 1 to 3. More specifically, whereas two peaks appear at retention times of 3.07 min (peak 1 ) and 3.79 min (peak 4) in the control samples, these product peaks are absent from the experimental samples as can be seen in Figures 1 to 3. The concentration profile and/or the absence of distinct product peaks from the experimental samples gives statistically relevant information as to the presence of malignancy in the tested individual.
  • Example 3 Determination of profiles of amino acids and amino acid related compounds in blood samples of cancer patients and healthy donors
  • Heparinized blood samples of patients were incubated for 2.5 h at 37 0 C in Eppendorf tubes without and with 100 ⁇ of Ukrain ® , diluted to 1 :5, 1 : 10, 1 : 100 or 1 : 1000, followed by a centrifugation step for 15 min at 1500 g in an Eppendorf microcentrifuge.
  • the such obtained plasma was deproteinized by admixing an equal volume of 1 M perchloric acid (HCIO 4 ).
  • Homotaurine (Sigma) was used as an internal standard.
  • the protein-free extracts obtained by this procedure were stored at -20 0 C and kept for subsequent analysis according to Example 1 .

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  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Immunology (AREA)
  • Engineering & Computer Science (AREA)
  • Cell Biology (AREA)
  • Chemical & Material Sciences (AREA)
  • Urology & Nephrology (AREA)
  • Biomedical Technology (AREA)
  • Molecular Biology (AREA)
  • Hematology (AREA)
  • Microbiology (AREA)
  • Biotechnology (AREA)
  • Oncology (AREA)
  • Hospice & Palliative Care (AREA)
  • Food Science & Technology (AREA)
  • Medicinal Chemistry (AREA)
  • Physics & Mathematics (AREA)
  • Analytical Chemistry (AREA)
  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • General Physics & Mathematics (AREA)
  • Pathology (AREA)
  • Investigating Or Analysing Biological Materials (AREA)

Abstract

La présente invention concerne un procédé de diagnostic ex vivo permettant la détection d'un développement oncogène malin ou d'un cancer chez un être humain, le procédé étant basé sur la détermination des modifications qualitatives et/ou quantitatives d'au moins un composé, de préférence d'un acide aminé ou d'un composé apparenté à cet acide aminé, présent dans un fluide corporel d'un individu malade par comparaison avec un témoin sain.
PCT/EP2007/002461 2006-03-20 2007-03-20 Procédé de diagnostic du cancer ex vivo WO2007107334A1 (fr)

Applications Claiming Priority (4)

Application Number Priority Date Filing Date Title
EP06005682 2006-03-20
EP06005682.7 2006-03-20
EP07001963 2007-01-30
EP07001963.3 2007-01-30

Publications (1)

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WO2007107334A1 true WO2007107334A1 (fr) 2007-09-27

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Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US9459255B2 (en) 2006-12-21 2016-10-04 Ajinomoto Co., Inc. Method of evaluating breast cancer, breast cancer-evaluating apparatus, breast cancer-evaluating method, breast cancer-evaluating system, breast cancer-evaluating program and recording medium
US9465031B2 (en) 2008-06-20 2016-10-11 Ajinomoto Co., Inc. Method of evaluating prostatic disease
US9599618B2 (en) 2006-12-21 2017-03-21 Ajinomoto Co., Inc. Method, apparatus, system, program, and computer-readable recording medium for evaluating colorectal cancer
JP2017198694A (ja) * 2008-02-06 2017-11-02 味の素株式会社 取得方法、胃癌評価装置、胃癌評価プログラムおよび胃癌評価システム

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4444890A (en) * 1982-02-05 1984-04-24 Burzynski Stanislaw R Testing procedure to aid diagnosis of cancer and evaluate the progress of cancer therapy
JPS61126472A (ja) * 1984-11-24 1986-06-13 Advance Res & Dev Co Ltd 診断方法
WO2006053680A1 (fr) * 2004-11-19 2006-05-26 Nowicky Wassili Procede permettant de diagnostiquer un cancer ex vivo

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4444890A (en) * 1982-02-05 1984-04-24 Burzynski Stanislaw R Testing procedure to aid diagnosis of cancer and evaluate the progress of cancer therapy
JPS61126472A (ja) * 1984-11-24 1986-06-13 Advance Res & Dev Co Ltd 診断方法
WO2006053680A1 (fr) * 2004-11-19 2006-05-26 Nowicky Wassili Procede permettant de diagnostiquer un cancer ex vivo

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BIOCHEMICAL MEDICINE AND METABOLIC BIOLOGY OCT 1987, vol. 38, no. 2, October 1987 (1987-10-01), pages 127 - 133, ISSN: 0885-4505 *
CURRENT SCIENCE (BANGALORE), vol. 84, no. 4, 25 February 2003 (2003-02-25), pages 551 - 557, ISSN: 0011-3891 *
DATABASE BIOSIS [online] BIOSCIENCES INFORMATION SERVICE, PHILADELPHIA, PA, US; 25 February 2003 (2003-02-25), VENKATAKRISHNA K ET AL: "HPLC-LIF for early detection of oral cancer.", XP002435061, Database accession no. PREV200400168160 *
DATABASE MEDLINE [online] US NATIONAL LIBRARY OF MEDICINE (NLM), BETHESDA, MD, US; October 1987 (1987-10-01), HIRAYAMA C ET AL: "Plasma amino acid patterns in hepatocellular carcinoma.", XP002435062, Database accession no. NLM2823852 *
NEFYODOV L I ET AL: "AMINO ACIDS AND THEIR DERIVATIVES IN BLOOD PLASMA OF PATIENTS WITH BREAST CANCER TREATED WITH UKRAIN. PART V", DRUGS UNDER EXPERIMENTAL AND CLINICAL RESEARCH, BIOSCIENCE EDIPRINT INC, XX, vol. 22, no. 3 - 5, 1996, pages 155 - 157, XP008060480, ISSN: 0378-6501 *
NEFYODOV L I ET AL: "AMINO ACIDS AND THEIR DERIVATIVES IN TUMOUR TISSUE FROM PATIENTS WITH BREAST CANCER TREATED WITH UKRAIN. PART VI", DRUGS UNDER EXPERIMENTAL AND CLINICAL RESEARCH, BIOSCIENCE EDIPRINT INC, XX, vol. 12, no. 3-5, 1996, pages 159 - 161, XP008060488, ISSN: 0378-6501 *
UGLYANITSA K N ET AL: "BIOCHEMICAL MECHANISMS OF THE ANTICANCER EFFECT OF UKRAIN IN THE TREATMENT OF CANCER OF THE URINARY BLADDER", INTERNATIONAL JOURNAL OF NUCLEAR MEDICINE AND BIOLOGY, PERGAMON PRESS, OXFORD, GB, vol. 29, no. 1, 2001, pages 79 - 84, XP008079186, ISSN: 0047-0740 *
XIAOHUI FAN ET AL: "Diagnosis of breast cancer using HPLC metabonomics fingerprints coupled with computational methods", ENGINEERING IN MEDICINE AND BIOLOGY SOCIETY, 2005. IEEE-EMBS 2005. 27TH ANNUAL INTERNATIONAL CONFERENCE OF THE SHANGHAI, CHINA 01-04 SEPT. 2005, PISCATAWAY, NJ, USA,IEEE, 1 September 2005 (2005-09-01), pages 6081 - 6084, XP010907223, ISBN: 0-7803-8741-4 *

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US9459255B2 (en) 2006-12-21 2016-10-04 Ajinomoto Co., Inc. Method of evaluating breast cancer, breast cancer-evaluating apparatus, breast cancer-evaluating method, breast cancer-evaluating system, breast cancer-evaluating program and recording medium
US9599618B2 (en) 2006-12-21 2017-03-21 Ajinomoto Co., Inc. Method, apparatus, system, program, and computer-readable recording medium for evaluating colorectal cancer
JP2017198694A (ja) * 2008-02-06 2017-11-02 味の素株式会社 取得方法、胃癌評価装置、胃癌評価プログラムおよび胃癌評価システム
US9465031B2 (en) 2008-06-20 2016-10-11 Ajinomoto Co., Inc. Method of evaluating prostatic disease

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