Tumor energy metabolism profiling

• US 10,458,987 B2
• Filed: 07/02/2014
• Issued: 10/29/2019
• Est. Priority Date: 07/04/2013
• Status: Active Grant

• Current Assignee: Universitat Heidelberg
• Original Assignee: Universitat Heidelberg
• Inventors: Roth, Wilfried, Gdynia, Georg, Sauer, Sven
• Primary Examiner(s): Yamasaki, Robert J
• Assistant Examiner(s): Constantine, Charles Zoltan
• Application Number:
  US14/898,456
• Publication Number:
  US 20160146816A1
• Time in Patent Office: 1,945 Days
• Field of Search: None
• 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 for a time sufficient to eliminate nutrition, drug and biopsy effects on the energy metabolism in the sample materials:

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

(c) determination of enzyme activities of at least one anaerobic key enzyme and at least one aerobic key enzyme of the energy metabolism of the cell homogenate or the subcellular fraction and determination of a quotient for each key enzyme of the enzyme activity determined in the sample material and the enzyme activity determined in the comparative sample material wherein the one or more anaerobic key enzyme is selected from the group consisting of malic enzyme (ME), lactate dehydrogenase (LDH), pyruvate kinase low affinity (PKLA), hexokinase and an acyl-CoA dehydrogenase and wherein the one or more aerobic key enzyme is selected from the group consisting of pyruvate kinase high affinity (PKHA) and cytochrome c oxidase (COX); and

(d) calculation of a ratio of the sum of the anaerobic key enzyme quotients and the sum of the aerobic key enzyme quotients using the quotients determined in step (c) in the sample material and the comparative sample material, wherein a decrease in the ratio of aerobic to anaerobic key enzyme quotients or increase in the ratio of the anaerobic to aerobic key enzyme quotients, when compared to a value for balanced anaerobic and aerobic metabolism, correlates with tumor progression, wherein the patient sample material is selected from the group consisting of tumor tissue slices, tumor cell mass and isolated tumor cells and the comparative sample material is selected from the group consisting of a tumor distant tissue slice and a material which in step (a) is incubated under conditions that up regulate the anaerobic energy metabolism; and

(e) selecting a method of treatment based on the calculated ratio.

2. The diagnostic method of claim 1, wherein at least two anaerobic key enzymes and at least one aerobic key enzyme are selected.

3. The diagnostic method of claim 1, wherein two anaerobic key enzymes and one aerobic key enzyme are selected, and wherein LDH and PKLA are selected as the anaerobic key enzymes and PKHA is selected as the aerobic key enzyme.

4. The diagnostic method of claim 1, said method performed in a well plate, wherein the reagents for the determination of enzyme activity are pre-deposited on walls of the wells of the well plate.

5. 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 wherein a 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 for a time sufficient to eliminate nutrition, drug and biopsy effects on the energy metabolism of the tissue slices;

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

(c) determination of enzyme activities of at least one anaerobic key enzyme and at least one aerobic key enzyme of the energy metabolism of the cell homogenate or the subcellular fraction in the tumor tissue and the tumor distant tissue and determination of a quotient for each key enzyme of the enzyme activity determined in the tumor tissue and the enzyme activity determined in the tumor distant tissue wherein the one or more anaerobic key enzyme is selected from the group consisting of malic enzyme (ME), lactate dehydrogenase (LDH), pyruvate kinase low affinity (PKLA), hexokinase and an acyl-CoA dehydrogenase; and

wherein the one or more aerobic key enzyme is selected from the group consisting of pyruvate kinase high affinity (PKHA) and cytochrome c oxidase (COX); and

(d) calculation of a ratio of the sum of the anaerobic key enzyme quotients and the sum of the aerobic key enzyme quotients using the quotients determined in step (c) in the tumor tissue and the tumor distant tissue, wherein a decrease in the ratio of aerobic to anaerobic key enzyme quotients or an increase in the ratio of the anaerobic to aerobic key enzyme quotients, when compared to a value for balanced anaerobic and aerobic metabolism, correlates with tumor progression; and

(e) selecting a methods of treatment based on the calculated ratio.

6. The diagnostic method of claim 5, wherein at least two anaerobic key enzymes and at least one aerobic key enzyme are selected.

7. The diagnostic method of claim 5, wherein two anaerobic key enzymes and one aerobic key enzyme are selected, and wherein LDH and PKLA are selected as the anaerobic key enzymes and PKHA is selected as the aerobic key enzyme.

8. The diagnostic method of claim 5, said method performed in a well plate, wherein the reagents for the determination of enzyme activity are pre-deposited on walls of the wells of the well plate.

9. 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 wherein a fresh sample material comprises the tissue slice from a biopsy material comprising the steps of

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

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

(c) preparation of a cell homogenate and subcellular fractions from the tissue slice of step (a),

(d) determination of enzyme activities of at least one anaerobic key enzyme and at least one aerobic key enzyme of the energy metabolism of the cell homogenate or a subcellular fraction in the tumor tissue sample and in the tumor tissue sample with the up regulated anaerobic energy metabolism and formation of a quotient for each key enzyme of the enzyme activity in the tumor tissue and the enzyme activity determined in the tumor tissue sample with the up regulated anaerobic energy metabolism wherein the one or more anaerobic key enzyme is selected from the group consisting of malic enzyme (ME), lactate dehydrogenase (LDH), pyruvate kinase low affinity (PKLA), hexokinase and an acyl-CoA dehydrogenase and wherein the one or more aerobic key enzyme is selected from the group consisting of pyruvate kinase high affinity (PKHA) and cytochrome c oxidase (COX); and

(e) calculation of a ratio of the sum of the anaerobic key enzyme quotients to and the sum of the aerobic key enzyme quotients using the quotients determined in step (d) in the tumor tissue and in the tumor tissue sample with the up regulated anaerobic energy metabolism, wherein a decrease in the ratio of aerobic to anaerobic or an increase in the ratio of anaerobic to aerobic key enzyme quotients, when compared to a value for balanced anaerobic and aerobic metabolism, correlates with tumor progression; and

(f) selecting a method of treatment based on the calculated ratio.

10. The diagnostic method of claim 9, wherein at least two anaerobic key enzymes and at least one aerobic key enzyme are selected.

11. The diagnostic method of claim 9, wherein two anaerobic key enzymes and one aerobic key enzyme are selected, and wherein LDH and PKLA are selected as the anaerobic key enzymes and PKHA is selected as the aerobic key enzyme.

12. The diagnostic method of claim 9, said method performed in a well plate, wherein the reagents for the determination of enzyme activity are pre-deposited on walls of the wells of the well plate.

13. 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 wherein a fresh sample material comprises a tumor cell mass comprising the steps of

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

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

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

(d) determination of enzyme activity of at least one anaerobic key enzyme and at least one aerobic key enzyme of the energy metabolism of the subcellular 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 for each key enzyme of the enzyme activity in the tumor cell mass and the enzyme activity determined in the tumor cell mass with the up regulated anaerobic energy metabolism wherein the one or more anaerobic key enzyme is selected from the group consisting of malic enzyme (ME), lactate dehydrogenase (LDH), pyruvate kinase low affinity (PKLA), hexokinase and an acyl-CoA dehydrogenase and wherein the one or more aerobic key enzyme is selected from the group consisting of pyruvate kinase high affinity (PKHA) and cytochrome c oxidase (COX); and

(e) calculation of a ratio of the sum of the anaerobic key enzyme quotients and the sum of the aerobic key enzyme quotients using the quotients determined in step (d) in the tumor cell mass of step (a) and in the tumor cell mass with the up regulated anaerobic energy metabolism, wherein a decrease in the ratio of aerobic to anaerobic key enzyme quotients or an increase in the ratio of the anaerobic to aerobic key enzyme quotients, when compared to value for balanced anaerobic and aerobic metabolism, correlates with tumor progression; and

(f) selecting a method of treatment based on the calculated ratio.

14. The diagnostic method of claim 13, wherein at least two anaerobic key enzymes and at least one aerobic key enzyme are selected.

15. The diagnostic method of claim 13, wherein two anaerobic key enzymes and one aerobic key enzyme are selected, and wherein LDH and PKLA are selected as the anaerobic key enzymes and PKHA is selected as the aerobic key enzyme.

16. The diagnostic method of claim 13, said method performed in a well plate, wherein the reagents for the determination of enzyme activity are pre-deposited on walls of the wells of the well plate.

17. 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 wherein a fresh sample material comprises isolated cells from a solid or liquid tumor comprising the steps of

a) isolation of the cells from the solid or liquid tumor sample taken from a patient, estimation of the number of isolated tumor cells, dilution of the cells and incubation of the cells in a cell culture medium for a time sufficient to eliminate 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 as described in step (a) under conditions that up regulate the anaerobic energy metabolism;

(c) preparation of subcellular fractions and a cell homogenate from the isolated tumor cells of steps (a) and (b);

(d) determination of enzyme activity of at least one anaerobic key enzyme and at least one aerobic key enzyme of the energy metabolism of the sub cellular fraction or the cell homogenate in the isolated tumor cells and in the isolated tumor cells with the up regulated anaerobic energy metabolism and determination of a quotient for each key enzyme of the enzyme activity in the isolated tumor cells of step (a) and the enzyme activity determined in the isolated tumor cells with the up regulated anaerobic energy metabolism wherein the one or more anaerobic key enzyme is selected from the group consisting of malic enzyme (ME), lactate dehydrogenase (LDH), pyruvate kinase low affinity (PKLA), hexokinase and an acyl-CoA dehydrogenase; and

wherein the one or more aerobic key enzyme is selected from the group consisting of pyruvate kinase high affinity (PKHA) and cytochrome c oxidase (COX); and

(e) calculation of a ratio of the sum of the anaerobic key enzyme quotients and the sum of the aerobic key enzyme quotients using the quotient determined in step (d) in the isolated tumor cells of step (a) and in the isolated tumor cells with the up regulated anaerobic energy metabolism, wherein a decrease in the ratio of aerobic to anaerobic or an increase in the ratio of the anaerobic to aerobic key enzyme quotients, when compared to a value for balanced anaerobic and aerobic metabolism, correlates with tumor progression; and

(f) selecting a method of treatment based on the calculated ratio.

18. The diagnostic method of claim 17, wherein at least two anaerobic key enzymes and at least one aerobic key enzymes are selected.

19. The diagnostic method of claim 17, said method performed in a well plate, wherein the reagents for the determination of enzyme activity are pre-deposited on walls of the wells of the well plate.

20. The diagnostic method of claim 17, wherein two anaerobic key enzymes and one aerobic key enzyme are selected, and wherein LDH and PKLA are selected as the anaerobic key enzymes and PKHA is selected as the aerobic key enzyme.

21. The diagnostic method of claim 17, said method performed in a well plate, wherein the reagents for the determination of enzyme activity are pre-deposited on walls of the wells of the well plate.

22. A method for predicting tumor prognosis comprising the likelihood of formation of metastases, relapse occurrence, and/or local recurrence and based thereon providing alternative therapeutic treatments said method comprising

(a) incubating material from a fresh tumor sample obtained from a patient and a comparative fresh sample material from the patient in a culture medium for a time sufficient to eliminate nutrition, drug and biopsy effects on the energy metabolism in the sample materials;

(b) preparing a cell homogenate and subcellular fractions from the sample materials of step (a);

(c) determining the enzyme activities of at least one anaerobic key enzyme and at least one aerobic key enzyme of the energy metabolism of the cell homogenate or the subcellular fraction in the patient'"'"'s fresh tumor sample and in the patient'"'"'s comparative fresh sample, and determining a quotient for each key enzyme of the enzyme activity determined in the patient'"'"'s tumor sample and the enzyme activity determined in the patient'"'"'s comparative sample;

wherein the one or more anaerobic key enzyme is selected from the group consisting of malic enzyme (ME), lactate dehydrogenase (LDH), pyruvate kinase low affinity (PKLA), hexokinase and an acyl-CoA dehydrogenase and wherein the one or more aerobic key enzyme is selected from the group consisting of pyruvate kinase high affinity (PKHA) and cytochrome c oxidase (COX); and

d) calculating a ratio of the sum of the anaerobic key enzyme quotients and the sum of the aerobic key enzyme quotients using the quotients for each key enzyme determined in step (c) in the sample material and the comparative sample material, wherein a decrease in the ratio of aerobic to anaerobic key enzyme quotients or an increase in the ratio of the anaerobic to aerobic key enzyme quotients, when compared to a value for balanced anaerobic and aerobic metabolism, correlates with tumor progression, where the tumor sample material is selected from the group consisting of tumor tissue slices, tumor cell mass and isolated tumor cells and the comparative sample material is selected from the group consisting of a slice of tissue from a distant tumor or a material which in step (a) is incubated under conditions that up-regulate the anaerobic energy metabolism; and

(e) selecting a method of treatment based on the calculated ratio.

23. The diagnostic method of claim 22, wherein at least two anaerobic key enzymes and at least one aerobic key enzyme are selected.

24. The diagnostic method of claim 22, wherein two anaerobic key enzymes and one aerobic key enzyme are selected, and wherein LDH and PKLA are selected as the anaerobic key enzymes and PKHA is selected as the aerobic key enzyme.