TUMOR ENERGY METABOLISM PROFILING

• US 20160146816A1
• Filed: 07/02/2014
• Published: 05/26/2016
• Est. Priority Date: 07/04/2013
• Status: Active Grant

• Current Assignee: Universitat Heidelberg
• Original Assignee: Universitat Heidelberg
• Inventors: Roth, Wilfried, SAUER, Sven, GDYNIA, Georg
• Granted Patent: US 10,458,987 B2
• Time in Patent Office: Days
• US Class Current: 1/1
• CPC Class Codes: C12Q 1/00 Measuring or testing processes involving enzymes, nucleic acids or microorganisms measuring or testing apparatus with condition measuring or sensing means, e.g. colony counters, C12M1/34; Compositions therefor; Processes of preparing such compositions
• CPC Class Codes: G01N 2333/90 Enzymes; Proenzymes
• CPC Class Codes: G01N 2333/90206 acting on the CH-CH group of donors (1.3)
• CPC Class Codes: G01N 2333/90216 acting on a heme group of donors (1.9)
• CPC Class Codes: G01N 2333/904 acting on CHOH groups as donors, e.g. glucose oxidase, lactate dehydrogenase (1.1)
• CPC Class Codes: G01N 2333/91215 with a definite EC number (2.7.1.-)
• CPC Class Codes: G01N 2800/50 Determining the risk of developing a disease
• CPC Class Codes: G01N 2800/52 Predicting or monitoring the response to treatment, e.g. for selection of therapy based on assay results in personalised medicine; Prognosis
• CPC Class Codes: G01N 2800/54 Determining the risk of relapse
• CPC Class Codes: G01N 2800/7028 Cancer
• CPC Class Codes: G01N 2800/7066 Metabolic pathways
• CPC Class Codes: G01N 33/573 for enzymes or isoenzymes
• CPC Class Codes: G01N 33/574 for cancer
• CPC Class Codes: G01N 33/57484 involving compounds serving as markers for tumor, cancer, neoplasia, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides, metabolites

Abstract

A diagnostic method for the prediction of tumor prognosis including the likelihood of formation of metastases or of relapse or local recurrence in tumor related diseases in a mammal and the provision of a therapy recommendation for a patient the enzyme activity of key enzymes of the energy metabolism is determined in fresh tumor tissue or fresh tumor cell mass after 24 hours incubation in a cell culture medium. Incubation reduces nutrition, drug and biopsy/surgery effects on the energy metabolism of the tissue slices or the cell mass. The quotients of enzyme activity of anaerobic enzymes are put in ratio to the enzyme activity of aerobic enzymes or vice versa. Said ratio can be taken into account for prognosis of metastasis and a therapy recommendation.

Claims

1. A diagnostic method for the prediction of tumor prognosis including the likelihood of formation of metastases, relapse occurrence, and/or local recurrence and the provision of a therapy recommendation comprising the steps of

(a) incubation of a tumor patient'"'"'s fresh sample material and a fresh comparative sample material from the patient in a culture medium to allow elimination of nutrition, drug and biopsy effects on the energy metabolism in the sample materials;

(b) preparation of a cell homogenate and sub cellular fractions from the sample materials of step (a);

(c) determination of enzyme activities of at least one anaerobic and at least one aerobic key enzyme of the energy metabolism of the cell homogenate or the subcellular fraction and determination of a quotient of the enzyme activity of an enzyme determined in the sample material and the comparative sample material; and

(d) calculation of the ratio of anaerobic to aerobic energy gain or aerobic to anaerobic energy gain based on the quotient determined in step (c) in the sample material and the comparative sample material, and wherein the patient sample material is selected from tissue slices, tumor cell mass and isolated cells and the comparative sample material is selected from a tumor distant tissue slice or a material which in step (a) is incubated under conditions to up regulate the anaerobic energy metabolism and is selected from a tumor tissue slice, tumor cell mass and isolated cells.

2. The diagnostic method according to claim 1 wherein the fresh sample material comprises tissue slices from a biopsy material comprising the steps of

(a) incubation of the tissue slices taken from a biopsy material of a tumor tissue and of a tumor distant tissue of a patient in a culture medium to allow elimination of nutrition, drug and biopsy effects on the energy metabolism of the tissue slices;

(b) preparation of a cell homogenate and sub cellular fractions from the tissue slices of step (a);

(c) determination of enzyme activities of at least one anaerobic and at least one aerobic key enzyme of the energy metabolism of the cell homogenate or the subcellular fraction in tumor tissue and tumor distant tissue and determination of a quotient of the enzyme activity of an enzyme determined in tumor tissue and tumor distant tissue; and

(d) calculation of the ratio of anaerobic to aerobic energy gain or aerobic to anaerobic energy gain based on the quotient determined in step (c) in tumor tissue and tumor distant tissue.

3. The diagnostic method according to claim 1 wherein the fresh sample material comprises a tissue slice from a biopsy material comprising the steps of

(a) incubation of the tissue slice taken from a biopsy material of a tumor tissue of a patient in a cell culture medium to allow elimination of drug, nutrition and biopsy effects on the energy metabolism of the tissue slices;

(b) incubation of second tissue slice from the same biopsy material under conditions to up regulate the anaerobic energy metabolism in the tissue slice;

(c) preparation of a cell homogenate and sub cellular fractions from the tissue slices of step (a),

(d) determination of enzyme activities of at least one anaerobic and at least one aerobic key enzyme of the energy metabolism of the cell homogenate or the sub cellular fraction in the tumor tissue and in the tumor tissue sample with the up regulated anaerobic energy metabolism and formation of a quotient of the enzyme activity of an enzyme in the tumor tissue and in the tumor tissue sample with the up regulated anaerobic energy metabolism; and

(e) calculation of the ratio of anaerobic to aerobic energy gain or aerobic to anaerobic energy gain based on the quotient determined in step (c) in tumor tissue and in the tumor tissue sample with the up regulated anaerobic energy metabolism.

4. The diagnostic method according to claim 1 wherein the fresh sample material comprises a tumor cell mass comprising the steps of

(a) incubation of a part of a tumor cell mass taken from a patient in a cell culture medium to allow elimination of drug, nutrition and biopsy effects on the energy metabolism;

(b) incubation of another part of the same tumor cell mass taken from the patient under conditions to up regulate the anaerobic energy metabolism;

(c) preparation of a cell homogenate and sub cellular fractions from the tumor cell mass of step (a),

(d) determination of enzyme activity of at least one anaerobic and at least one aerobic key enzyme of the energy metabolism of the sub cellular fraction or the cell homogenate in the tumor cell mass and in the tumor cell mass with the up regulated anaerobic energy metabolism and determination of a quotient of the enzyme activity of an enzyme in the tumor cell mass and in the tumor cell mass with the up regulated anaerobic energy metabolism; and

(e) calculation of the ratio of anaerobic to aerobic energy gain or aerobic to anaerobic energy gain based on the quotient determined in step (c) in the tumor cell mass of step (a) and in the tumor cell mass with the up regulated anaerobic energy metabolism.

5. The diagnostic method according to claim 1 wherein the fresh sample material comprises isolated cells from a solid or liquid tumor comprising the steps of

(a) isolation of the cells from a solid or liquid tumor sample taken from a patient, estimation of the number of isolated cells, dilution of these cells and incubation of these cells in a cell culture medium to allow elimination of drug, nutrition and biopsy effects on the energy metabolism and to allow proliferation of the cells;

(b) incubation of the same amount of cells that were isolated and treated like described in (a) under conditions to up regulate the anaerobic energy metabolism;

(c) preparation of sub cellular fractions and a cell homogenate from the isolated cells of step (a) and (b);

(d) determination of enzyme activity of at least one anaerobic and at least one aerobic key enzyme of the energy metabolism of the sub cellular fraction or the cell homogenate in the isolated cells and in the isolated cells with the up regulated anaerobic energy metabolism and determination of a quotient of the enzyme activity of an enzyme in the isolated cells of (a) and in the isolated cells with the up regulated anaerobic energy metabolism; and

(e) calculation of the ratio of anaerobic to aerobic energy gain or aerobic to anaerobic energy gain based on the quotient determined in step (d) in the isolated cells of step (a) and in the isolated cells with the up regulated anaerobic energy metabolism.

6. The diagnostic method of claim 1, wherein at least one anaerobic enzyme and at least one aerobic enzyme of the key enzymes of the energy metabolism of the cell are selected.

7. The diagnostic method of claim 6, wherein the key enzymes of the energy metabolism of the cell are selected of one or more of malic enzyme (ME), lactate dehydrogenase (LDH), pyruvate kinase low affinity (PKLA or dimeric PK-M2), hexokinase and an acyl-CoA dehydrogenase and of one or more of the enzymes selected from pyruvate kinase high affinity (PKHA;

tetrameric PK-M2) and cytochrome c oxidase (COX).

8. The diagnostic method of claim 1, wherein the reagents for the determination of enzyme activity are pre-deposited at the walls of the wells of a well plate.

9. Use of the quotient of the enzyme activity of at least one anaerobic enzyme and at least one aerobic enzyme of the key enzymes of the energy metabolism determined (i) in fresh tumor tissue and as comparison in fresh tumor distant tissue or (ii) in tumor tissue or tumor cells and as comparison under anoxic condition as a marker or a biomarker for the prediction of tumor prognosis including the likelihood of formation of metastases, relapse occurrence, and/or local recurrence wherein said quotient is determined by

(a) incubation of a tumor patient'"'"'s fresh sample material and a fresh comparative sample material from the patient in a culture medium to allow elimination of nutrition, drug and biopsy effects on the energy metabolism in the sample materials;

and

(b) preparation of a cell homogenate and sub cellular fractions from the sample materials of step (a); and

(c) determination of enzyme activities of at least one anaerobic and at least one aerobic key enzyme of the energy metabolism of the cell homogenate or the subcellular fraction, said quotient representing the enzyme activity of an enzyme determined in the sample material and the comparative sample material;

wherein the patient sample material is selected from tissue slices, tumor cell mass and isolated cells and the comparative sample material is selected from a tumor distant tissue slice or a material which in step (a) is incubated under conditions to up regulate the anaerobic energy metabolism and is selected from a tumor tissue slice, tumor cell mass and isolated cells.

10. Use according to claim 9 wherein one or more enzymes are selected from the group of malic enzyme (ME), lactate dehydrogenase (LDH), pyruvate kinase low affinity (PKLA or dimeric PK-M2), hexokinase and an acyl-CoA dehydrogenase as anaerobic enzyme and one or more of the group selected from pyruvate kinase high affinity (PKHA;

tetrameric PK-M2) and cytochrome c oxidase (COX) as aerobic enzyme.