Methods and compositions for the diagnosis of cancer susceptibilities and defective DNA repair mechanisms and treatment thereof

• US 20030188326A1
• Filed: 06/06/2002
• Published: 10/02/2003
• Est. Priority Date: 11/03/2000
• Status: Abandoned Application

• Current Assignee: Dana-Farber Cancer Institute, Oregon Health & Science University
• Original Assignee: Dana-Farber Cancer Institute
• Inventors: Grompe, Markus, Taniguchi, Toshiyasu, Fox, Edward A., Timmers, Cynthia, DʼAndrea, Alan D.
• Application Number:
  US10/165,099
• Publication Number:
  US 20030188326A1
• Time in Patent Office: Days
• US Class Current: 800/8
• CPC Class Codes: A01K 2217/05 Animals comprising random inserted nucleic acids (transgenic)
• CPC Class Codes: A01K 2217/075 inducing loss of function, i.e. knock out
• CPC Class Codes: C07K 14/47 from mammals
• CPC Class Codes: C07K 16/18 against material from animals or humans
• CPC Class Codes: C12Q 1/025 for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics antimicrobial activity C12Q1/18
• CPC Class Codes: C12Q 1/6886 for cancer immunoassay for cancer G01N33/574
• CPC Class Codes: C12Q 2600/156 Polymorphic or mutational markers
• CPC Class Codes: C12Q 2600/158 Expression markers
• CPC Class Codes: G01N 2333/47 Assays involving proteins of known structure or function as defined in the subgroups
• CPC Class Codes: G01N 2500/00 Screening for compounds of potential therapeutic value
• 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 33/5011 for testing antineoplastic activity
• CPC Class Codes: G01N 33/5091 for testing the pathological state of an organism
• 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
• CPC Class Codes: G01N 33/6893 related to diseases not provided for elsewhere

Abstract

Methods and compositions for the diagnosis of cancer susceptibilities, defective DNA repair mechanisms and treatments thereof are provided. Among sequences provided here, the FANCD2 gene has been identified, and probes and primers are provided for screening patients in genetic-based tests and for diagnosing Fanconi Anemia and cancer. The FANCD2 gene can be targeted in vivo for preparing experimental mouse models for use in screening new therapeutic agents for treating conditions involving defective DNA repair. The FANCD2 polypeptide has been sequenced and has been shown to exist in two isoforms identified as FANCD2-S and the monoubiquinated FANCD-L form. Antibodies including polyclonal and monoclonal antibodies have been prepared that distinguish the two isoforms and have been used in diagnostic tests to determine whether a subject has an intact Fanconi Anemia/BRCA pathway.

Claims

1. A method of diagnosing or determining if a patient has cancer or is at increased risk of cancer, the method comprising testing a Fanconi Anemia/BRCA pathway gene for the presence of a cancer-associated defect, wherein said presence of one or more cancer-associated defects is indicative of cancer or an increased risk of cancer in said patient.

2. The method according to claim 1, wherein said cancer is breast, ovarian, or prostate cancer.

3. The method according to claim 1, wherein said cancer-associated defect results in a reduction in the ratio of FANC D2-L relative to FANC D2-S as compared to said ratio in a patient without one or more cancer-associated defects in a Fanconi Anemia/BRCA pathway gene.

4. A method of diagnosing or determining if a patient has cancer or is at increased risk of cancer, the method comprising testing a Fanconi Anemia/BRCA pathway protein for the presence of a cancer-associated defect, wherein said presence of a cancer-associated defect is indicative of cancer or an increased risk of cancer in said patient.

5. The method of claim 4, wherein said cancer is breast, ovarian, or prostate cancer.

6. A method of diagnosing or determining if a patient is at increased risk of developing cancer, comprising the steps of:

(a) providing a tissue sample from said patient;

(b) inducing DNA damage in the cells of said tissue sample; and

(c) assaying for the presence of FANC D2-S and FANC D2-L proteins in said cells;

wherein a reduction in the ratio of FANC D2-L to FANC D2-S is indicative that said patient is at increased risk of developing cancer.

7. The method of claim 6, wherein said cancer is breast, ovarian, or prostate cancer.

8. The method of claim 6, wherein said patient has no known cancer-associated defects in the BRCA-1 or BRCA-2 genes.

9. The method of claim 6, wherein said patient has one or more cancer-associated defects in the BRCA-1 or BRCA-2 genes.

10. The method of claim 6, wherein a plurality of said tissue samples are distributed in an array.

11. A method of determining if a patient has cancer, or is at increased risk of developing cancer, wherein the patient has no known cancer causing defect in the BRCA 1 or BRCA-2 genes, said method comprising the steps of:

(a) providing a DNA sample from said patient;

(b) amplifying the FANC D2 gene from said patient with the FANC D2 gene-specific polynucleotide primers of SEQ ID NOs;

115-186;

(c) sequencing the amplified FANC D2 gene; and

(d) comparing the FANC D2 gene sequence from said patient to a reference FANC D2 gene sequence, where a discrepancy between the two gene sequences indicates the presence of a cancer-associated defect;

wherein the presence of one or more cancer-associated defects indicates said patient has cancer or is at an increased risk of developing cancer.

12. The method of claim 11, wherein said cancer is breast, ovarian, or prostate cancer.

13. The method of claim 11, wherein said patient has no known cancer-associated defects in the BRCA-1 or FANC-D1/BRCA-2 genes.

14. The method of claim 11, wherein said patient has one or more cancer-associated defects in the BRCA-1 or FANC-D1/BRCA-2 genes.

15. The method of claim 11, wherein a plurality of said DNA samples are distributed on a microarray.

16. A method of screening for a chemosensitizing agent, said method comprising the steps of:

(a) providing a potential inhibitor of the Fanconi Anemia/BRCA pathway;

(b) providing a tumor cell line that is resistant to one or more anti-neoplastic agents;

(c) contacting said tumor cell line and said potential inhibitor of the Fanconi Anemia/BRCA pathway and said one or more anti-neoplastic agents; and

(d) measuring the growth rate of said tumor cell line in the presence of said inhibitor of the Fanconi Anemia/BRCA pathway and said anti-neoplastic agent;

wherein a reduced growth rate of the tumor cell line, relative to cells of the tumor cell line in the presence of the anti-neoplastic agent and the absence of said inhibitor of the Fanconi Anemia/BRCA pathway, is indicative that the potential inhibitor is a chemosensitizing agent.

17. The method of claim 16, wherein said potential inhibitors of the Fanconi Anemia/BRCA pathway are screened on a microarray, wherein the microarray contains addresses containing one or more cells that are resistant to one or more anti-neoplastic agents.

18. The method according to claim 16, wherein said potential inhibitor of the Fanconi Anemia/BRCA pathway is an inhibitor of the ubiquitination of the FANC D2 protein.

19. The method according to claim 16, wherein said anti-neoplastic agent is cisplatin.

20. The method according to claim 16, wherein said tumor cell line is an ovary cancer cell line.

21. A method of treating a patient having a cancer, wherein the cancer is resistant to a anti-neoplastic agent, comprising the step of administering a therapeutically effective amount of an inhibitor of the Fanconi Anemia/BRCA pathway together with said anti-neoplastic agent.

22. The method according to claim 21, wherein the said anti-neoplastic agent is cisplatin.

23. The method according to claim 21, wherein said potential inhibitor of the Fanconi Anemia/BRCA pathway is an inhibitor of the ubiquitination of the FANC D2 protein.

24. The method according to claim 21, wherein said tumor cell line is an ovary cancer cell line.

25. A method for screening for a cancer therapeutic, the method comprising the steps of:

(a) providing one or more cells containing a Fanconi Anemia/BRCA pathway gene having one or more cancer associated defects;

(b) growing said cells in the presence of a potential cancer therapeutic; and

(c) determining the rate of growth of said cells in the presence of said potential cancer therapeutic relative to the rate of growth of equivalent cells grown in the absence of said potential cancer therapeutic;

wherein a reduced rate of growth of said cells in the presence of said potential cancer therapeutic, relative to the rate of growth of equivalent cells grown in the absence of said potential cancer therapeutic, indicates that the potential cancer then is a cancer therapeutic.

26. The method of claim 26, wherein said cells containing a Fanconi Anemia/BRCA pathway gene having one or more cancer associated defects are distributed in a array.

27. A method of predicting the efficacy of a therapeutic agent in a cancer patient, comprising the steps of:

(a) providing a tissue sample from said cancer patient who is being treated with said therapeutic agent;

(b) inducing DNA damage in the cells of said tissue sample;

(c) detecting the presence of FANC D2-L protein in said cells;

wherein the presence of FANC D2-L is indicative of a reduced efficacy of said therapeutic agent in said cancer patient.

28. The method of claim 27, wherein said therapeutic agent is cisplatin.

29. A method of determining resistance of tumor cells to an anti-neoplastic agent, comprising the steps of:

(a) providing a tissue sample from a patient who is being treated with an anti-neoplastic agent;

(b) inducing DNA damage in the cells of said tissue sample; and

(c) determining the methylation state of a Fanconi Anemia/BRCA pathway gene;

wherein methylation of a Fanconi Anemia/BRCA gene is indicative of resistance of the tumor cells to an anti-neoplastic agent.

30. The method according to claim 29, wherein said Fanconi Anemia/BRCA gene is the FANC F gene.

31. The method according to claim 29, wherein said anti-neoplastic agent is cisplatin.

32. A kit for detecting defects in the FANC D2 gene, comprising a polynucleotide primer pair specific for the FANC D2 gene, a reference FANC D2 gene sequence and packaging materials therefore.

33. A kit for detecting the presence of FANC D2-L, comprising a FANC D2-L-specific antibody and packaging materials therefore.

34. A kit for determining the methylation state of a Fanconi Anemia/BRCA pathway gene, comprising FANC D2 polynucleotide primer pairs and probes, a control unmethylated reference FANC D2 gene sequence and packaging materials therefore.

35. A kit for screening for a chemosensitizing agent, comprising a tumor cell line that is resistant to one or more anti-neoplastic agents and packaging materials therefore.

36. The kit of claim 35, wherein said tumor cell line is an ovary tumor cell line.

37. The kit according to claim 36, wherein said ovary tumor cell line is a cisplatin resistant ovary tumor cell line.

38. The kit according to claim 36, wherein said anti-neoplastic agent is cisplatin.

39. A microarray containing one or more nucleic acid sequences from one or more Fanconi Anemia/BRCA pathway genes.

40. The microarray of claim 39, wherein the genes are selected from the group consisting of:

ATM, FANC A, FANC B, FANC C, FANC D1, FANC D2, FANC E, FANC F and FANC G.

41. A method of determining if a patient has cancer, or is at increased risk of developing cancer, said method comprising the steps of:

(a) providing the microarray of claim 39;

(b) providing a nucleic acid sample from said patient;

(c) hybridizing said nucleic acid sample to said nucleic acid sequences from the Fanconi Anemia/BRCA pathway on said microarray; and

(d) detecting the presence of mutations in the Fanconi Anemia/BRCA pathway genes in the nucleic acid sample from said patient;

wherein said detecting the presence of mutations is indicative of a patient who has cancer, or is at increased risk of developing cancer.