U.S. patent application number 09/200629 was filed with the patent office on 2002-08-01 for antibodies against md2 protein.
Invention is credited to CORDON-CARDO, CARLOS, FINLAY, CATHY A., LEVINE, ARNOLD J..
Application Number | 20020102721 09/200629 |
Document ID | / |
Family ID | 27533802 |
Filed Date | 2002-08-01 |
United States Patent
Application |
20020102721 |
Kind Code |
A1 |
LEVINE, ARNOLD J. ; et
al. |
August 1, 2002 |
ANTIBODIES AGAINST MD2 PROTEIN
Abstract
The invention provides a method of diagnosing cancer by
determining the expression level or gene amplification of p53 and
dm2, whereby an elevated level of either p53 or dm2 or both p53 and
dm2 indicates a cancer diagnosis. Furthermore, the invention
provides a method of predicting the progress of cancer by
determining the expression level or gene amplification of p53 and
dm2, whereby an elevated level of either p53 or dm2 or both 53 and
dm2 indicates a poor prognosis.
Inventors: |
LEVINE, ARNOLD J.; (NEW
YORK, NY) ; FINLAY, CATHY A.; (CHAPEL HILL, NC)
; CORDON-CARDO, CARLOS; (NEW YORK, NY) |
Correspondence
Address: |
IMCLONE SYSTEMS INCORPORATED
180 VARICK STREET
NEW YORK
NY
10014
|
Family ID: |
27533802 |
Appl. No.: |
09/200629 |
Filed: |
November 30, 1998 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
09200629 |
Nov 30, 1998 |
|
|
|
08362590 |
Mar 31, 1995 |
|
|
|
08362590 |
Mar 31, 1995 |
|
|
|
PCT/US93/06163 |
Jun 28, 1993 |
|
|
|
09200629 |
Nov 30, 1998 |
|
|
|
08018649 |
Feb 17, 1993 |
|
|
|
08018649 |
Feb 17, 1993 |
|
|
|
07904766 |
Jun 26, 1992 |
|
|
|
07904766 |
Jun 26, 1992 |
|
|
|
07730185 |
Jul 12, 1991 |
|
|
|
07730185 |
Jul 12, 1991 |
|
|
|
07543963 |
Jun 27, 1990 |
|
|
|
Current U.S.
Class: |
435/320.1 |
Current CPC
Class: |
Y10S 436/813 20130101;
C07K 14/475 20130101; C07K 14/82 20130101; C12Q 2600/118 20130101;
C07K 16/32 20130101; C12Q 2600/136 20130101; G01N 33/57496
20130101; C12Q 1/6886 20130101; G01N 33/5748 20130101; G01N 2333/82
20130101; C07K 14/4746 20130101; C07K 16/22 20130101 |
Class at
Publication: |
435/320.1 |
International
Class: |
C12N 015/63 |
Claims
We claim:
1. A method of diagnosing cancer by determining the level of p53
and dm2 in a biological sample, whereby an elevated level of either
p53 or dm2 or both p53 and dm2 indicates a cancer diagnosis.
2. A method of predicting the progress of cancer by determining the
level of p53 and dm2 in a biological sample, whereby an elevated
level of either p53 or dm2 or both p53 and dm2 indicates a poor
prognosis.
3. A method of classifying a biological sample into one of three
groups, the method comprising determining whether the level of
either p53 or dm2 in the sample is abnormally elevated, whereby the
first group comprises no abnormal elevation of either the level of
p53 or dm2, the second group comprises abnormal elevation of the
level of p53 and no abnormal elevation of the level of dm2 or
abnormal elevation of the level of dm2 and no abnormal elevation of
the level of p53, and the third group comprises abnormal elevation
of the level of both p53 and dm2.
4. A method of assessing a subject's prognosis by obtaining a
biological sample from the subject, classifying the sample's group
using the method of claim 3, whereby, of the three groups, the
third group indicates the worst prognosis.
5. A method of assessing a subject's prognosis by obtaining a
biological sample from the subject, determining the sample's group
using the method of claim 3, whereby, of the three groups, the
first group indicates the best prognosis.
6. A method according to claim 3, wherein the level of dm2 gene
amplification or expression is determined by using probes.
7. A method according to claim 6, wherein the probes are
antibodies.
8. A method according to claim 7, wherein the antibodies are
monoclonal.
9. A method according to claim 8, wherein the antibodies are
selected from the group consisting of claims 14-18.
10. A monoclonal antibody of claim 27, 28, 29, 30 or 31 bound to a
toxin.
11. A method of treating a patient with a tumor by contacting the
tumor with an antibody of claim 10 under conditions such that the
antibody binds to the tumor.
12. A monoclonal antibody of claim 27, 28, 29, 30 or 31 labeled
with a detectable marker.
13. A method for imaging tumors which comprises contacting the
tumor to be imaged with an anti-dm2 antibody of claim 12, under
conditions such that the antibody binds to the tumor and detecting
the antibody bound thereto, thereby imaging the tumor.
14. A method of detecting in a biological sample cancer cells or
cells at risk of becoming cancerous or pre-cancerous, wherein the
cells contain at least one normal p53 allele, the method comprising
determining whether the level of dm2 in the biological sample is
abnormally elevated in comparison with the level of dm2 expression,
whereby an abnormally elevated level of dm2 in the biological
sample indicates cancer cells or cells at risk of becoming
cancerous or pre-cancerous.
15. A method according to claim 14, wherein the elevated level is
determined by immunohistochemical staining of the biological
sample.
16. A method according to claim 14, wherein the level of dm2 gene
amplification or expression is determined by using probes.
17. A method according to claim 16, wherein the probes are nucleic
acid probes.
18. A method according to claim 16, wherein the probes are
antibodies.
19. A method according to claim 18, wherein the antibodies are
monoclonal.
20. A p53/dm2 complex.
21. A method according to claim 19, wherein the monoclonal antibody
is produced by hybridoma 3G5, deposited under ATCC Accession No. HB
11182.
22. A method according to claim 19, wherein the monoclonal antibody
is produced by hybridoma 4B11, deposited under ATCC Accession No.
HB 11183.
23. A method according to claim 19, wherein the monoclonal antibody
is produced by hybridoma 2A10, deposited under ATCC Accession No.
HB 11184.
24. A method according to claim 19, wherein the monoclonal antibody
is produced by hybridoma 2A9, deposited under ATCC Accession No. HB
11185.
25. A method according to claim 19, wherein the monoclonal antibody
is produced by hybridoma 4B2, deposited under ATCC Accession No. HB
11186.
26. A method according to claim 19, wherein the monoclonal antibody
recognizes the same epitope or epitopes as those recognized by a
monoclonal antibody produced by a hybridoma selected from the group
consisting of hybridoma 3G5, deposited under ATCC Accession No. HB
11182; hybridoma 4B11, deposited under ATCC Accession No. HB 11183;
hybridoma 2A10, deposited under ATCC Accession No. HB 11184;
hybridoma 2A9, deposited under ATCC Accession No. HB 11185; and
hybridoma 4B2, deposited under ATCC Accession No. HB 11186.
27. A method according to claim 19, wherein the monoclonal antibody
competes with the same epitope or epitopes as those recognized by a
monoclonal antibody produced by a hybridoma selected from the group
consisting of hybridoma 3G5, deposited under ATCC Accession No. HB
11182; hybridoma 4B11, deposited under ATCC Accession No. HB 11183;
hybridoma 2A10, deposited under ATCC Accession No. HB 11184;
hybridoma 2A9, deposited under ATCC Accession No. HB 11185; and
hybridoma 4B2, deposited under ATCC Accession No. HB 11186.
28. A method according to claim 27, wherein the competing
monoclonal antibody blocks between 50-100% of the epitope or
epitopes recognized by the monoclonal antibody produced by a
hybridoma selected from the group consisting of hybridoma 3G5,
deposited under ATCC Accession No. HB 11182; hybridoma 4B11,
deposited under ATCC Accession No. HB 11183; hybridoma 2A10,
deposited under ATCC Accession No. HB 11184; hybridoma 2A9,
deposited under ATCC Accession No. HB 11185; and hybridoma 4B2,
deposited under ATCC Accession No. HB 11186.
29. The monoclonal antibody produced by hybridoma 3G5, deposited
under ATCC Accession No. HB 11182.
30. The monoclonal antibody produced by hybridoma 4B11, deposited
under ATCC Accession No. HB 11183.
31. The monoclonal antibody produced by hybridoma 2A10, deposited
under ATCC Accession No. HB 11184.
32. The monoclonal antibody produced by hybridoma 2A9, deposited
under ATCC Accession No. HB 11185.
33. The monoclonal antibody produced by hybridoma 4B2, deposited
under ATCC Accession No. HB 11186.
34. A monoclonal antibody that recognizes the same epitope or
epitopes as those recognized by a monoclonal antibody produced by a
hybridoma selected from the group consisting of hybridoma 3G5,
deposited under ATCC Accession No. HB 11182; hybridoma 4B11,
deposited under ATCC Accession No. HB 11183; hybridoma 2A10,
deposited under ATCC Accession No. HB 11184; hybridoma 2A9,
deposited under ATCC Accession No. HB 11185; and hybridoma 4B2,
deposited under ATCC Accession No. HB 11186.
35. A monoclonal antibody that competes with the same epitope or
epitopes as those recognized by a monoclonal antibody produced by a
hybridoma selected from the group consisting of hybridoma 3G5,
deposited under ATCC Accession No. HB 11182; hybridoma 4B11,
deposited under ATCC Accession No. HB 11183; hybridoma 2A10,
deposited under ATCC Accession No. HB 11184; hybridoma 2A9,
deposited under ATCC Accession No. HB 11185; and hybridoma 4B2,
deposited under ATCC Accession No. HB 11186.
36. A monoclonal antibody according to claim 35 that blocks between
50-100% of the epitope or epitopes recognized by the monoclonal
antibody produced by a hybridoma selected from the group consisting
of hybridoma 3G5, deposited under ATCC Accession No. HB 11182;
hybridoma 4B11, deposited under ATCC Accession No. HB 11183;
hybridoma 2A10, deposited under ATCC Accession No. HB 11184;
hybridoma 2A9, deposited under ATCC Accession No. HB 11185; and
hybridoma 4B2, deposited under ATCC Accession No. HB 11186.
37. A hybridoma selected from the group consisting of hybridoma
3G5, deposited under ATCC Accession No. HB 11182; hybridoma 4B11,
deposited under ATCC Accession No. HB 11183; hybridoma 2A10,
deposited under ATCC Accession No. HB 11184; hybridoma 2A9,
deposited under ATCC Accession No. HB 11185; and hybridoma 4B2,
deposited under ATCC Accession No. HB 11186.
38. Any epitope recognized by a monoclonal antibody selected from
the group of monoclonal antibodies of claims 29-33.
39. An epitope recognized by a monoclonal antibody of claim 31.
40. A diagnostic kit comprising: (a) a container comprising an
anti-p53 antibody that recognizes a p53 protein; and (b) a
container comprising an anti-dm2 antibody that recognizes a dm2
protein.
41. A diagnostic kit of claim 40, wherein the antibodies are
labeled.
42. A diagnostic kit of claim 40, further comprising: (a) a labeled
antibody that recognizes the anti-p53 antibody; and (b) a labeled
antibody that recognizes the anti-dm2 antibody.
Description
[0001] This application is a continuation-in-part of PCT
Application No. ______, filed Jun. 25, 1993, which is a
continuation-in-part of U.S. Ser. No. 08/018,649, filed Feb. 17,
1993, which is a continuation-in-part of U.S. Ser. No. 07/904,766,
filed Jun. 26, 1992, which in turn is a continuation-in-part of
U.S. Ser. No. 07/730,185, filed Jul. 12, 1991, which in turn is a
continuation-in-part of U.S. Ser. No. 07/543,963 filed Jun. 27,
1990, all of which are incorporated herein by reference.
BACKGROUND OF THE INVENTION
[0002] Mutations of proto-oncogenes in somatic cells are
increasingly being recognized as significant in the induction of
human cancers. Some examples of oncogenes formed by such mutations
include: neu, fes, fos, myc, myb, fms, Ha-ras, and Ki-ras. The
mutations that convert proto-oncogenes to oncogenes are often point
mutations. Much needs to be learned in order to understand how
oncogenes and their expression products function to transform
normal cells to cancer cells.
[0003] Oncogenes are generally believed to act in a dominant
fashion. This is generally considered to mean that the conversion
of a proto-oncogene to an oncogene results in the acquisition of a
new function, i.e., enhancing transformation.
[0004] A different type of mutation associated with cancer occurs
when a tumor suppressor gene is altered in a way that causes the
product of the gene to lose its tumor suppressor function. An
example of such a tumor suppressor gene is the retinoblastoma
susceptibility gene, Rb. Tumor suppressor genes are sometimes
called recessive oncogenes, although, strictly speaking, the
products of tumor suppressor genes do not contribute to tumor
formation. The phenotype is recessive since, when both alleles are
mutated, the absence of a tumor suppressor gene results in an
enhancement of tumorigenesis.
[0005] A gene product that exhibits some properties of both a
dominant and a recessive oncogene is the 53kd phosphoprotein, p53.
Evidence is growing that mutations in the p53 gene is associated
with a large number of many types of cancers. For example, Iggo et
al., Lancet 335, 675-679 (1990) has expressed the opinion that p53
is the most common proto-oncogene to undergo mutation in lung
cancers.
[0006] Much of what is known about p53 has been derived from
studying the effect of transfecting wild-type and mutant murine p53
in rat embryo fibroblast cells. This work has been reviewed by
Levine et al., "The P53 Proto-Oncogene And Its Product," in Common
Mechanisms Of Transformation By Small DNA Tumor Viruses, L.
Villarreal, ed., American Society for Microbiology, Chapter 2
(1989); Hinds et al., ibid, Chapter 7; and Levine, BioEssays 12,
60-66 (1990).
[0007] The p53 gene appears to be involved in transcriptional
control (Fields, S. & Jang, S. K. (1990) Science 249,
1046-1049; Raycroft, L., Wu, H. & Lozano, C. (1990) Science
249, 1049-1051; and Levine, A. J., Momand, J. & Finlay, C. A.
(1991) Nature 351, 453-456) and may act as a regulatory check point
in the cell cycle, arresting cells in the G-1 phase (Martinez, J.,
Georgoff, I. & Levine, A. J. (1991) Genes Dev. 5, 151-159;
Hupp, T. R., Meek, D. W., Midgley, C. A. & Lane, D. P. (1992)
Cell 71, 875-886; and Yin, Y., Tainsky, M. A., Bischoff, F. Z.,
Strong, C. C. & Wahl, E. M. (1992) Cell 70, 937-948). Genetic
alterations of the p53 gene, such as intragenic mutations,
homozygous deletions, and structural rearrangements, are frequent
events in human cancer (Vogelstein, B. & Kinzler, K. (1992)
Cell 70, 523-526; Baker, S. J. et al. (1990) Cancer Res 50,
7717-7722; Mori, N. et al. (1989) Cancer Res 49, 5130-5135; Lee, J.
H. et al. (1990) Cancer Res 50, 2724-2728; Varley, J. M. et al.
(1991) Oncogene 6, 413-421; Presti, J. C. et al. (1991) Cancer Res
51, 5405; Dalbagni, G., et al. (1993) Diagnostic Molecular
Pathology 2, 4-13). These altered patterns of p53 either reduce or
inhibit the activity of functional homotetramer units (Stenger, J.
E., et al. (1992) Mol Carcinog 5, 102-106; Sturzbecher, H. W., et
al. (1992) Oncogene 7, 1513-1523). Mutant p53 proteins have a
prolonged half-life and retarded degradation, yielding accumulation
of inactive complexes and self-aggregatory molecules in the nuclei
of tumor cells (Sturzbecher, H. W. et al. (1987) Oncogene 1,
201-211; Halevy, O. et al. (1989) Mol Cell Biol 9, 3385-3392),
[0008] In humans, germ-line mutations of the p53 gene have been
characterized in members of families affected with the Li-Fraumeni
syndrome, a rare autosomal dominant trait that predisposes these
individuals to develop a variety of tumors, including soft tissue
sarcomas (Li, F. P. & Fraumeni, J. F. (1969) Ann Intern Med 71,
747-752; Malkin, D. et al. (1990) N. Engl. J. Med. 250, 1233-1238).
More recently, p53 germ-line mutations were also detected in cancer
patients with no apparent family history of cancer (Toguchida, J.
et al. (1992) N. Engl. J. Med. 326, 1301-1308), as well as a subset
of patients presenting with a second primary neoplasm (Malkin D.,
et al. (1992) New Eng J Med 326, 1309-1315). In addition, somatic
mutations of the p53 gene have been reported to occur in soft
tissue sarcomas (Stratton, M. R., et al. (1990) Oncogene 5,
1297-1301; Mulligan, L. M., et al. (1990) Proc Natl Acad Sci USA
87, 5863-5867; Toguchida, J. et al. (1992) Cancer Res 52,
6194-6199; Drobnjak, M. et al. (1993) Submitted; Latres, E., et al.
(1993) Submitted).
[0009] Another gene that has been identified as having oncogenic
potential is the murine double minute-2 (mdm2) gene (Fakharzadeh et
al., (1991) The EMBO Journal 10(6):1565-1569). The sequences of the
murine and human mdm2 genes and proteins are known (Fakharzadeh et
al., The EMBO Journal 10(6):1565-1569 (1991); Oliner, J. D. et al.
(1992) Nature 358:80-83; and Cahilly-Snyder et al., Somatic Cell
and Molecular Genetics, 13(3):235-244 (1987)). The sequence is
evolutionarily conserved among species including mouse, rat,
hamster and human genomes (Cahilly-Snyder et al., Somatic Cell and
Molecular Genetics, 13(3):235-244 (1987)).
[0010] The mdm2 gene is also referred to in the literature as MDM2,
MDM2 and hdm2 (the human homologs) and mdm2 (murine). The mdm2 gene
product, which is a 90 kD phosphoprotein, is referred to in the
literature as p90, which refers to both murine and human homologs,
and MDM2, which is the human homolog. The p90 protein is described
in applicants' related publication, Levine et al., International
Application No. PCT/US91/04608, filed Jun. 27, 1991. Where dm2 is
used throughout this application, it is meant to encompasses the
various terms in the literature for the mdm2 gene and protein,
including homologs among all species.
[0011] There is evidence for MDM2 amplification and MDM2 (gene
product) overexpression in both osteo- and soft tissue sarcomas
(Oliner, J. D. et al. (1992) Nature 358, 80-83; Ladanyi, M. et al.
(1993) Cancer Res 53, 16-18; Leach, F. S. et al. (1993) Cancer Res
53, 2231-2234).
[0012] The human homolog of the mdm2 gene, called the hdm2 gene or
MDM2 or MDM2, has been cloned and mapped to the long arm of
chromosome 12 (12q13-14) (Oliner et al. 1992. Amplification of a
gene encoding a p53-associated protein in human sarcomas. Nature
358:80-83). This region contains two genes, SAS and GLI, previously
found to be amplified in osteo- and soft tissue sarcomas. The SAS
gene codes for a protein of unidentified function. It was isolated
from a malignant fibrous histiocytoma (MFH), and was shown to be
amplified in MFH and liposarcomas (Turc-Carel, C. et al. (1986)
Cancer Genet Cytogenet 23, 291-299; Meltzer, P. S. et al. (1991)
Cell Growth Diff 2, 495-501). The GLI gene codes for a DNA-binding
zinc finger protein. Even though it was originally isolated from a
glioblastoma, it has also been reported to be amplified in a
rhabdomyosarcoma and an osteosarcoma (Kinzler, K. et al.(1984)
Science 236, 70-73).
[0013] Prior to the present invention, it was known that mutated
p53 is associated with cancer. Futhermore, it was known prior to
the present invention that overexpression of mdm2 is associated
with tumors. However, there has been no disclosure prior to the
present invention of the relationship between altered p53 and dm2
genes and their altered patterns of expression in cells and how to
utilize this relationship to diagnose as well as to determine the
clinical relevance or prognoses of cancer patients. An objective of
the subject invention is to utilize the relationship between p53
and dm2 overexpressed or amplified genes to diagnose cancer as well
as to determine the prognoses of cancer patients.
SUMMARY OF THE INVENTION
[0014] The subject invention provides a method of diagnosing cancer
by determining the level of p53 and dm2 in a biological sample,
whereby an elevated level of either p53 or dm2 or both p53 and dm2
indicates a cancer diagnosis.
[0015] A further objective of the invention has been met by
providing a method of predicting the progress of cancer by
determining the level of p53 and dm2 in a biological sample,
whereby an elevated level of either p53 or dm2 or both 53 and dm2
indicates a poor prognosis.
[0016] The invention further provides a method of classifying a
biological sample into one of three groups, the method comprising
determining whether the level of either p53 or dm2 in the sample is
abnormally elevated, whereby the first group comprises no abnormal
elevation of either the level of p53 or dm2, the second group
comprises abnormal elevation of the level of p53 and no abnormal
elevation of the level of dm2 or abnormal elevation of the level of
dm2 and no abnormal elevation of the level of p53, and the third
group comprises abnormal elevation of the level of both p53 and
dm2.
[0017] The invention further provides a method of detecting in a
biological sample cancer cells or cells at risk of becoming
cancerous or pre-cancerous, wherein the cells contain at least one
normal p53 allele. The method comprises determining whether the
level of dm2 in the biological sample is abnormally elevated,
whereby an elevated level of dm2 in the biological sample in
comparison to normal biological samples indicates cancer cells or
cells at risk of becoming cancerous or pre-cancerous.
[0018] The invention also provides an isolated p53/dm2 protein
complex and antibodies to the dm2 protein.
BRIEF DESCRIPTION OF THE FIGURES
[0019] FIG. 1: This figure indicates epitopes of the p90 protein
that react with the anti-p90 monoclonal antibodies of the
invention. The anti-p90 monoclonal antibodies produced by
hybridomas 1F5, 6C10, 1D6, 4B2, 2E12, 3F3, 3G5, 3F8, 6H7, 2A9, 3G9,
1D11, 2A10, 1G2, 4B11 and 5B10 are indicated beneath the p90 amino
acid epitope map at locations of the map indicating the epitopes
with which such antibodies react.
[0020] FIG. 2: This graph shows the overall survival of 211
patients with soft tissue sarcomas over a period of 90 months.
[0021] FIG. 3: This graph shows p53/mdm2 elevated levels and
survival in the group of 211 patients (see FIG. 2) with soft tissue
sarcomas. The phenotypic categories shown in the graph are as
follows: Group A: dm2-/p53- (neither dm2 nor p53 had elevated
levels); Group B: dm2+/p53- and dm2-/p53+; and Group C:
dm2+/p53+(elevated levels of both dm2 and p53).
[0022] FIG. 4: This photograph illustrates immunostaining patterns
using anti-dm2 and anti-p53 antibodies. The control group refers to
the top two slides. The top left slide shows a staining pattern of
a 3T3-Balb/c cell line, which is dm2-. The top right slide shows a
staining pattern of 3T3-DM cell line, which is dm2+. The middle two
slides show a staining pattern of human tumor tissue sections taken
from the same patient. The middle left slide shows a p53-staining
pattern, and the middle right slide shows a dm2+staining pattern
(corresponding to the Group B subset p53-/dm2+ subset described
below). The bottom two slides show a staining pattern of human
tumor tissue sections taken from a different patient. The bottom
left slide shows a p53+ staining pattern, and the bottom right
slide shows a dm2+ staining pattern (corresponding to Group C
described below).
DETAILED DESCRIPTION OF THE INVENTION
[0023] DEFINITIONS:
[0024] p53:
[0025] For the purposes of the present specification, the term
"wild-type" p53 means the nucleotide or amino acid sequence
reported by Matlashewski et al., EMBO J. 13, 3257-3262 (1984);
Zakut-Houri et al., EMBO J. 4, 1251-1255 (1985); and Lamb and
Crawford, Mol. Cell. Biol. 5, 1379-1385 (1986). The sequences are
available from GenBank. Wild-type p53 includes a proline/arginine
polymorphism at amino acid 72 and the corresponding nucleotide
polymorphism.
[0026] Mutations of wild-type p53 genes and proteins indicate
pre-cancer and cancer states. A pre-cancer cell is a cell that
typically has one normal p53 allele and one mutated p53 allele. For
example, the mutation may be a point mutation. In a cancer cell,
both p53 alleles are usually mutated. For example, one mutation may
be a point mutation, and the other mutation may be a deletion of
all or a significant part of the p53 gene.
[0027] dm2:
[0028] The dm2 of this invention refers to a family of proteins
that includes a phosphoprotein of 90 kD (p90), its fragments or
products, including proteins p85 (85 kD), p76 (76 kD), p74 (74 kD)
and p58-57 (58 kD and 57 kD) (p57 is the murine equivalent to the
rat p58), and homologs or analogs thereof. The p53 protein
co-immunoprecipitates with the dm2 protein. The dm2 protein
includes the known, sequenced murine double minute 2 (mdm2) 90 kD
phosphoprotein. Furthermore, the term "dm2" refers to genes
encoding for the family of dm2 proteins described above. The
sequence of the mdm2 gene and protein is disclosed by Fakharzadeh
et al., The EMBO Journal 10(6):1565-1569 (1991); and Cahilly-Snyder
et al., Somatic Cell and Molecular Genetics, 13(3):235-244 (1987)).
Sequences homologous to the p90 dm2 are present in the genomes of
several species including human ("hdm2"). The human gene has been
sequenced and has a molecular weight of approximately 90 kD
(Oliner, J. D. et al. (1992) Nature 358:80-83), as well as rat,
mouse and hamster. In a preferred embodiment, the dm2 gene and
protein are human.
[0029] dm2 proteins of approximately 90 kD clearly, and p58 most
likely, binds to p53. 3T3DM cells derived from Balb/c 3T3
overproduce the five mdm2 protein species in response to the
amplified mdm2 gene copy.
[0030] Elevated Levels of dm2 and/or p53 (dm2+ and/or P53+):
[0031] For the purposes of this specification, elevated levels of
dm2 and/or p53 in a cell may indicate dm2 or p53 gene
amplification, or of dm2 or p53 protein product overexpression or
accumulation in a biological sample, such as a cell. Elevated
levels of dm2 or p53 protein are a measure of total dm2 or p53
protein in a biological sample, preferably a cell, whether free
protein or in a complex. In some cases, the dm2 protein elevated
levels in the absence of dm2 amplification indicates the formation
of heterodimers/heterotetramers between dm2 and mutated p53
products. Mutant p53 proteins have a prolonged half-life and
retarded degradation, and therefore accumulate in the cell nuclei.
p53 missense mutations represent the majority, at least about 85%,
of the p53 mutations detectable by immunochemistry. However, in
some cases, wild-type p53 genes and proteins are detected at
elevated levels in tumor tissue.
[0032] An elevated level of dm2 and/or p53 in a biological sample
in comparison to normal biological samples indicates a cancer cell
or cell at risk of becoming cancerous or pre-cancerous. A
biological sample may include but is not limited to, tissue
extracts, cell samples or biological fluids such as lymph, blood or
urine.
[0033] The subject invention provides a method of classifying a
biological sample into one of three groups, the method comprising
determining whether the level of either p53 or dm2, or both p53 and
dm2, in the sample is abnormally elevated. The first group, Group
A, comprises no abnormal elevation of either the level of p53 or
dm2 (p53-/dm2-); the second group, Group B, comprises abnormal
elevation of the level of dm2 and no abnormal elevation of the
level of p53 (p53-/dm2+), or abnormal elevation of the level of p53
and no abnormal elevation of the level of dm2 (p53+/dm2-); and the
third group, Group C, comprises abnormal elevation of the level of
both p53 and dm2 (p53+/dm2+).
[0034] The invention demonstrates that classifying altered patterns
of dm2 and p53 expression is clinically significant for the
diagnosis and prediction of the clinical outcome of patients with
various types of cancer. Such cancers include sarcomas, carcinomas
and leukemias or lymphomas. Particularly, such cancers include
sarcomas, such as soft tissue sarcomas and osteogenic sarcomas. In
another embodiment, the cancers include bladder cancers. Other
embodiments include, but are not limited to, colorectal, lung,
ovarian, cervical, adrenal cortex, bone, breast, brain, chronic
myelocytic leukemia, and chronic myelogenous leukemia. Therefore,
the invention provides a method of assessing a subject's prognosis
by obtaining a biological sample from pre-cancer tissue or a tumor
of the subject, and determining to which of the three groups, A, B,
or C, the biological sample belongs. Of the three categories, the
third group indicates the worst prognosis and the first group
indicates the best prognosis.
[0035] Group C (p53+/dm2+):
[0036] The invention provides a method for detecting a cancer cell
or cell at risk of becoming cancerous or pre-cancerous, wherein the
cell contains at least one mutant p53 allele, by determining
whether elevated levels of dm2 are present.
[0037] The invention unexpectedly demonstrates that elevated levels
of dm2 protein act synergistically with elevated levels of p53
protein, including mutant as well as overexpressed wild-type forms
of p53 protein, which may be detected as elevated levels of p53,
i.e., the p53+/dm2+ group (see FIG. 4), resulting in
clinicopathological variables of poor prognosis, including survival
and tumor progression. Of the three groups, Group C indicates the
worst prognosis. Example 2 and FIG. 3 show that the immunological
detection of abnormally elevated levels of dm2 and p53 in the same
tumor sections occurred in a group with very poor survival when
compared to Groups A (no p53 or dm2 elevated levels) and B
(elevated levels of only one of these proteins).
[0038] This is unexpected because, as discovered by the inventors,
dm2 protein inactivates p53 transcriptional activity, and therefore
one might not have expected elevated levels of dm2 protein in a
cell with elevated levels of p53 protein.
[0039] Group B (p53-/dm2+ and p53+/dm2-)
[0040] Group B contains two subsets, normal levels of p53 with
elevated levels of dm2 (p53-/dm2+) and elevated levels of p53 with
normal levels of dm2 (p53+/dm2-). Of the three groups, Group B
indicates a poorer prognosis than Group A, but better prognosis
than Group C.
[0041] Subset p53+/dm2- of Group B:
[0042] The detection of elevated levels of p53, such as mutated or
overexpressed wild-type p53 genes and proteins, i.e. the p53+/dm2-
group, indicate pre-cancer and cancer states. Mutant forms of p53
can inactivate the wild-type p53 function, and cells containing
mutant p53 genes and proteins have an enhanced tumorigenic
potential. Furthermore, elevated levels of wild-type p53 protein
may contribute to poor prognosis. DNA damage in cells or binding to
viral oncogene products stabilize wild-type p53 protein and
increase its concentration.
[0043] Subset p53-/dm2+ of Group B:
[0044] The invention unexpectedly demonstrates that elevated levels
of dm2 confer a similar property on cells, such as such as BALB/c
3T3 cells (3T3 DM cells) or pre-cancer or cancer cells, as does
elevated levels of p53. In both cases, the cells that express high
levels of dm2 or p53 protein gain an enhanced tumorigenic potential
in animals.
[0045] The invention provides that, with regard to the p53-/dm2+
group, despite the presence of normal levels of wild-type p53
allele or alleles in a cell, elevated levels of dm2 enhance the
tumorigenic potential of the cell.
[0046] The invention provides a method of detecting a cancer cell
or cell at risk of becoming cancerous or pre-cancerous wherein the
cell contains two normal p53 alleles, i.e. wild-type p53, or normal
levels of p53, comprising determining whether the level of dm2 in
the biological sample is abnormally elevated, meaning elevated in
comparison with the level of dm2 in normal biological samples. (see
FIG. 4).
[0047] Group A (p53-/dm.sup.2-)
[0048] Group A comprises normal levels of both p53 and dm2.
Precancer and cancer cells in this group are most likely due to
factors other than elevated levels of p53 and/or dm2. Of the three
groups, Group A indicates the best prognosis.
[0049] Detection of Elevated Levels of dm2 or p53:
[0050] Amplification:
[0051] Amplification of the dm2 or p53 gene may be detected using
methods well known in the art, such as nucleic acid probe
technology. High copy numbers of DNA may, for example, be detected
using Southern blotting, and increased amounts of RNA may be
detected using Northern blotting (see George, D. L. and Powers, V.
E., Cell 24:117-123 (1981); George, D. L. et al. EMBO J.,
4:1199-1203 (1985); and Singh, L. and Jones, K. W. Nucleic Acids
Res., 12:5627-5638 (1984).
[0052] Overexpression:
[0053] Levels of dm2 and p53 protein in the biological sample are
detected by methods well known in the art, such as by using
anti-dm2 and anti-p53 antibodies and immunohistochemical staining.
A positive nuclear staining when using immunohistochemistry
indicate elevated levels of p53 or dm2 or p53/dm2 complexes.
Overexpressed p53 proteins are associated with p53 gene mutations.
In one embodiment of the invention, dm2 and p53 nuclear
overexpression in tumors is classified into one of three groups by
estimating the percentage of tumor cell nuclei staining: (a)
negative (<20%), (b) heterogeneous (20-70%), (c) homogeneous
(>70%).
[0054] In another embodiment of the invention, the elevated level
of dm2 is detected by a method indicating that a pre-cancer or
cancer state is 2-100 times that of normal biological samples, such
as cells. In a preferred embodiment, the elevated level is a level
5-50 times that of normal biological samples, such as cells.
[0055] Polyclonal and monoclonal antibodies may be prepared by
methods known in the art. Antibodies of this invention include
recombinant polyclonal or monoclonal Fab fragments prepared in
accordance with the method of Huse et al., Science 246:1275-1281
(1989). See Campbell, "Monoclonal Antibody Technology, The
Production and Characterization of Rodent and Human Hybridomas" in
Burdon et al., Eds, Laboratory Techniques in Biochemistry and
Molecular Biology, Volume 13, Elsevier Science Publishers,
Amsterdam (1985). Methods for preparing polyclonal and monoclonal
antibodies that exhibit specificity toward single amino acid
differences between oncogenes are described by McCormick et al. in
U.S. Pat. No. 4,798,787.
[0056] Briefly, polyclonal antibodies may be produced by injecting
a host mammal, such as a rabbit, mouse, rat, or goat, with the p53
protein or a fragment thereof capable of producing antibodies that
distinguish between mutant p53 and wild-type p53. The peptide or
peptide fragment injected may contain the wild-type sequence or the
mutant sequence. Sera from the mammal are extracted and screened to
obtain polyclonal antibodies that are specific to the peptide or
peptide fragment. The same method may be applied to dm2
proteins.
[0057] In order to produce monoclonal antibodies, a host mammal is
inoculated with a peptide or peptide fragment as described above,
and then boosted. Spleens are collected from inoculated mammals a
few days after the final boost. Cell suspensions from the spleens
are fused with a tumor cell in accordance with the general method
described by Kohler and Milstein in Nature 256, 495-497 (1975). In
order to be useful, a peptide fragment must contain sufficient
amino acid residues to define the epitope of the p53 or dm2
molecule being detected.
[0058] If the fragment is too short to be immunogenic, it may be
conjugated to a carrier molecule. Some suitable carrier molecules
include keyhole limpet hemocyanin and bovine serum albumen.
Conjugation may be carried out by methods known in the art. One
such method is to combine a cysteine residue of the fragment with a
cysteine residue on the carrier molecule.
[0059] The peptide fragments may be synthesized by methods known in
the art. Some suitable methods are described by Stuart and Young in
"Solid Phase Peptide Synthesis," Second Edition, Pierce Chemical
Company (1984).
[0060] Suitable antibodies for the co-immunoprecipitation of p53
and dm2 include PAb421 and Ab2. PAb421 recognizes the
carboxy-terminus of p53 from various species, including human,
mouse and rat p53, and is described by Harlow et al. in the Journal
of Virology 39, 861-869 (1981). Ab2 is specific for the
amino-terminus of human p53, and is available from Oncogene
Science, Inc. of Manhassett, N.Y. The dm2 protein does not
immunoprecipitate when REF cells that do not express p53 are
treated in the same way with the same antibodies.
[0061] The immunoprecipitates are recovered by centrifugation.
Following centrifugation or elution from the column, dm2 may be
separated from the p53/dm2 complex by means of SDS PAGE. The single
band at 90kD is cut and sequenced. This invention also provides an
isolated dm2/p53 complex, obtained by the coimmunoprecipitating of
dm2 with p53 from a variety of transformed cells.
[0062] Another method for purifying dm2 is that generally described
by Aebersold et al., Proc. Natl. Acad. Sci. USA 84, 6970-6974
(1987).
[0063] This invention provides hybridomas expressing monoclonal
antibodies against the dm2 (p90) gene product. Certain of these
hybridomas are deposited at the American Type Culture Collection
(ATCC): 3G5 ATCC Accession No. HB 11182); 4B11 (ATCC Accession No.
HB 11183); 2A10 (ATCC Accession No. HB 11184); 2A9 (ATCC Accession
No. 11185); and 4B2 (ATCC Accession No. HB 11186). Hybridomas 3G5,
4B11, 2A10, 2A9 and 4B2, were deposited pursuant to, and in
satisfaction of, the requirements of the Budapest Treaty on the
International Recognition of the Deposit of Microorganisms for the
Purposes of Patent Procedure with the American Type Culture
Collection (ATCC), 12301 Parklawn Drive, Rockville, Md. 20852 under
ATCC Accession Nos. HB 11182, HB 11183, HB 11184, HB 11185 and HB
11186, respectively.
[0064] Assays for Determining and Modifying the Level of dm2 in
Cells
[0065] The level of dm2 and/or p53 in cells is determined by assays
known in the art capable of recognizing amplified or overexpressed
dm2 and/or p53 genes or proteins.
[0066] A variety of assays are available for detecting proteins
with labeled antibodies. In a one-step assay, the target molecule,
if it is present, is immobilized and incubated with a labeled
antibody. The labeled antibody binds to the immobilized target
molecule. After washing to remove unbound molecules, the sample is
assayed for the presence of the label.
[0067] In a two-step assay, immobilized target molecule is
incubated with an unlabeled antibody. The target molecule-unlabeled
antibody complex, if present, is then bound to a second, labeled
antibody that is specific for the unlabeled antibody. The sample is
washed and assayed for the presence of the label, as described
above.
[0068] Labeled anti-dm2 antibodies may be used to detect dm2 using
imaging methods. One method for imaging comprises contacting the
tumor cell to be imaged with an anti-dm2 antibody labeled with a
detectable marker. The method is performed under conditions such
that the labeled antibody binds to the dm2. The antibody bound to
the dm2 is detected, thereby imaging and detecting the dm2.
[0069] The choice of marker used to label the antibodies will vary
depending upon the application. However, the choice of marker is
readily determinable to one skilled in the art. These labeled
antibodies may be used in immunoassays as well as in histological
applications to detect the presence of tumors. The labeled
antibodies may be polyclonal or monoclonal. In a preferred
embodiment, the antibodies are monoclonal, and are the antibodies
deposited with the ATCC listed above.
[0070] In preferred embodiments of the invention, the label may be
a radioactive atom, an enzyme, or a chromophoric moiety. Some
examples of radioactive atoms include P.sup.32, I.sup.125, H.sup.3,
and C.sup.14. Some examples of enzymes include horseradish
peroxidase, alkaline phosphatase, beta-galactosidase, and
glucose-6-phosphate dehydrogenase. Some examples of chromophoric
moieties include fluorescein and rhodamine. The antibodies may be
conjugated to these labels by methods known in the art. For
example, enzymes and chromophoric molecules may be conjugated to
the antibodies by means of coupling agents, such as dialdehydes,
carbodiimides, dimaleimides, and the like. Alternatively,
conjugation may occur through a ligand-receptor pair. Some suitable
ligand-receptor pairs include, for example, biotin-avidin
or--streptavidin, and antibody-antigen.
[0071] Antibodies against the dm2 protein may be used to detect
elevated levels of the dm2 protein in cells or tissue. In one
embodiment, antibodies that are directed at epitopes of dm2 may be
used in situ using immunohistopathology techniques. These methods
have diagnostic use where there is a high risk of tissue becoming
cancerous, such as in polyps or atypical breast tissue, or in tumor
cells where there is a greater risk of metastasis or
recurrence.
[0072] The hybridomas 1F5, 6C10, 1D6, 4B2, 2E12, 3F3, 3G5, 3F8,
6H7, 2A9, 3G9, 1D11, 2A10, 1G2, 4B11 and 5B10, some of which were
deposited with the ATCC (see above), produce monoclonal antibodies
against dm2 protein epitopes (see FIG. 1). The monoclonal
antibodies react with both murine and human protein epitopes, as
well as with other species, due to the homologous dm2 sequences
conserved between the species.
[0073] In another embodiment, cancer cells may release dm2 and
therefore increase the amount of dm2 in the blood or lymph of a
subject. Therefore, assays such as immunoassays may be used to
detect normal levels or levels of dm2 above normal in cells or
bodily fluids.
[0074] Screening for Therapeutics to Block dm2 Binding to Wild-Type
p53:
[0075] Assays may be used to screen for therapeutics to inhibit dm2
binding to wild-type p53. The subject invention discloses methods
for selecting a therapeutic which forms a complex with dm2 with
sufficient affinity to prevent the deleterious binding of dm2 to
wild-type p53. The methods include various assays, including
competitive assays where the dm2 is immobilized to a support, and
is contacted with both wild-type p53 and a labeled therapeutic
either simultaneously or in either consecutive order, and
determining whether the therapeutic effectively competes with the
wild-type p53 in a manner sufficient to prevent binding of dm2 to
wild-type p53. In another embodiment, the wild-type p53 is labeled
and the therapeutic is unlabeled. In a further embodiment, the p53
is immobilized to a support, and is contacted with both labeled dm2
and a therapeutic (or unlabeled dm2 and a labeled therapeutic), and
determining whether the amount of dm2 bound to the p53 is reduced
in comparison to the assay without the therapeutic added. The dm2
may be labeled with the anti-dm2 antibodies of the subject
invention.
[0076] In one embodiment, the method comprises:
[0077] a) contacting a solid support with a predetermined amount of
dm2 under conditions permitting dm2 to attach to the surface of the
support, such as by using an anti-dm2 antibody to tether the dm2 to
the solid support; b) removing any dm2 which is not bound to the
support; c) contacting the solid support to which the dm2 is bound
with wild-type p53 under conditions such that the wild-type p53
binds to the bound dm2 and forms a complex therewith; d) removing
any unbound p53; e) contacting the dm2/p53 complex so formed with a
predetermined amount of the sample labeled with a detectable marker
under conditions such that the labeled potential therapeutic
present in the sample competes with the wild-type p53 for binding
to the bound dm2; f) quantitatively determining the concentration
of labeled potential therapeutic not bound to the solid support;
and g) thereby quantitatively determining the concentration of
potential therapeutic in the sample that specifically binds to dm2
to block dm2 binding to wild-type p53.
[0078] The choice of solid support may be readily determined by one
skilled in the art. In one preferred method, the solid support is a
bead formed of an inert polymer, in another the solid support is a
microwell. The markers used in the above-described method are a
matter of choice to one skilled in the art. It is preferred that
the detectable marker is an enzyme, a paramagnetic ion, biotin, a
fluorophore, a chromophore, a heavy metal, or a radioisotope. More
preferably, the marker is an enzyme, and most preferably, the
enzyme is horseradish peroxidase or alkaline phosphatase.
[0079] A further embodiment of this method is wherein the potential
therapeutic is labeled with an enzyme and step (f) comprises
removing the labeled potential therapeutic which was not bound to
the solid support and contacting it with specific substrate to the
enzyme under conditions such that the enzyme reacts with the
substrate to form a detectable product.
[0080] The subject invention also provides a method of treating
cancer in mammals including mice, rats, hamsters and humans, which
comprises blocking the deleterious binding of dm2 to wild-type p53.
One embodiment of this method comprises blocking the binding of dm2
to wild-type p53 by contacting the dm2 with a sufficient amount of
anti-dm2 antibody. Another embodiment of this method comprises
blocking the binding of dm2 to wild-type p53 by contacting the dm2
with an excess of an anti-idiotypic p53 antibody. In another
embodiment, dm2 anti-idiotypic antibodies may be administered which
do not block the transcription promoter of p53. In another method
for treating tumors, gene therapy may be used to replace the
amplified dm2 genes with a normal number of dm2 genes to regulate
elevated levels of p53. In a further method of treating tumors,
anti-sense gene therapy is used to replace the amplified dm2 gene
with an antisense dm2 gene.
[0081] Methods for determining relative binding affinities may be
conducted by methods known in the art. For example, a method for
determining whether a p53 protein binds to hsc70 is described by
Finlay et al. in Mol. and Cell. Biol. 8, 531-539 (1988) and by
Hinds et al. in Mol. and Cell. Biol., 7, 2863-2869 (1987). The
method described in these papers involves co-immunoprecipitation
experiments with anti-p53 and anti-hsc70 antibodies.
EXAMPLES
Example 1
[0082] Isolation of cDNA Clones.
[0083] A .lambda.gt11 cDNA library prepared from HeLa cells was
screened with the mouse mdm2 cDNA as probe under reduced
stringency. The cDNA inserts were isolated from positive phages and
subcloned into the Bluescript vector for further characterization.
A full length cDNA containing the entire coding region was
reconstructed from two overlapping clones and completely sequenced
using the method of Sanger (Sanger et al. 1977. DNA sequencing with
chain-terminating inhibitors. Proc. Natl. Acad. Sci. USA
14:5463-5467).
[0084] Generation of Monoclonal Antibodies Against hdm2.
[0085] A cDNA clone obtained from the library screening contained
the N-terminal coding region of the hdm2 coding region, truncated
at the first methionine initiation codon. This cDNA was recombined
with the full length hdm2 cDNA to obtain a coding region without
leader sequence and the first methionine. This coding sequence was
then inserted into the pQEll vector (Quiagen) to obtain a complete
open reading frame with 6 histidine residues fused to the
N-terminus of hdm2. The expression plasmid was then introduced into
E. coli, the Histidine-hdm2 fusion protein was purified by
Ni-.sup.2+-NTA-agarose (Quiagen) column chromatography. The major
protein species in the purified preparation has a mobility similar
to in vitro translated hdm2.
[0086] Balb/c mice were immunized with the E. coli produced hdm2
protein. Hybridomas were prepared using standard procedures, and
screened by enzyme-linked immunosorbent assay and
immunoprecipitation of in vitro translated hdm2 protein. Stable
clones were established by three rounds of cloning.
[0087] Isolation of Human dm2 cDNA.
[0088] A .lambda.gt11 library constructed from HeLa cells was
screened using the mouse mdm2 cDNA under conditions of reduced
stringency. A total of 14 positive clones were isolated and the
CDNA inserts subcloned into the Bluescript vector for further
analysis. Preliminary restriction mapping and partial sequencing
showed that they represent partial clones for the human dm2 cDNA
(Fakharzadeh, S. S. et al. 1991. Tumorigenic potential associated
with enhanced expression of a gene that is amplified in a mouse
tumor cell line. EMBO J. 10:1565-1569). A full length coding region
was constructed from two overlapping cDNA clones and sequenced. The
DNA sequence of this CDNA clone, designated hdm2, is similar to the
published hdm2 sequence (Oliner, J. D. et al. 1992. Amplification
of a gene encoding a p53-associated protein in human sarcomas.
Nature 358:80-83), with complete identitity within the coding
region and a few differences in the noncoding regions. The fact
that these two CDNA clones were obtained from two very different
sources (HeLa cell vs. colon carcinoma), yet have identical coding
sequences, suggests that they may represent the wild-type hdm2
coding sequence or a systematic mutation is present in different
cancer cells.
Example 2
[0089] A cohort of 211 soft tissue sarcomas of adults that were
clinically and pathologically well characterized were analyzed in
this example. In a subgroup of 73 patients, tumor and normal tissue
specimens were available. This group of 73 patients was used to
address the molecular and biological considerations of the study.
However, clinicopathological correlations were conducted using
immunohistochemistry and information regarding 211 patients with
soft tissue sarcomas obtained from a frequently updated database of
clinical and pathological information available for patients at
Memorial Sloan-Kettering Cancer Center.
[0090] TISSUE.
[0091] Of the cohort of 211 adult patients affected with soft
tissue sarcomas (STS) used for this example, the tumor lesions
analyzed included 71 liposarcomas, 53 leiomyosarcomas, 22 malignant
fibrous histiocytoma, 15 fibrosarcomas, 15 peripheral nerve sheath
tumors (PNST), 13 synovial sarcomas, 4 rhabdomyosarcomas, and 18
undifferentiated sarcomas. The majority of STS analyzed presented
as primary tumors (n=129), while the remaining lesions were either
recurrent (n=39) or metastatic (n=41). Presentation status in two
cases was unknown. Of the 211 STS analyzed, 169 cases were
classified as high grade sarcomas, while 39 tumors were considered
to be low grade lesions. The grade of 3 cases was unknown. The
median and mean follow-up times for this cohort of patients were 29
and 34 months, respectively. Complete follow-up data was available
for 209 of the 211 patients.
[0092] Tumor specimens were embedded in cryopreservative solution
(OCT compound, Miles Laboratories, Elkhart, Ind.), snap frozen in
isopentane and stored at -70.degree. C. Representative
hematoxylin-eosin stained sections of each block were examined
microscopically to confirm the presence of tumor, as well as to
evaluate percentage of tumor cells comprising these lesions and
extent of tumor necrosis. Adjacent tumor and normal tissue
specimens were also collected for molecular genetic assays in 73 of
211 cases (see below). These tissue samples were immediately frozen
after surgical removal and stored at -70.degree. C. prior to DNA
extraction.
[0093] Monoclonal Antibodies and Immunohistochemistry.
[0094] A panel of mouse monoclonal antibodies to the p90 gene
encoded gene product were used for the present study. Antibody 4B2
detects an epitope located in the amino-terminal region. Antibodies
2A9 and 2A10 identify two distinct epitopes in the central portion
of p90. Antibody 4B11 recognizes a sequence located in the
carboxy-terminal region of p90. Three mouse monoclonal antibodies
detecting different epitopes on p53 proteins were used for the
present study. Anti-p53 antibody PAbl80l (Ab-2, Oncogene Science,
Manhasset, N.Y.) recognizes an epitope located between amino acids
(aa) 32 to 79 of both wild-type and mutant human p53 proteins
(Banks, L. et al. (1986) Eur J Biochem 159, 529-534). Antibody
PAb240 (Ab-3, Oncogene Science) recognizes a conformational epitope
located between aa 156 to 335 characteristic of certain mutant p53
products (Gannon, J. V. et al. (1990) EMBO J 9, 1595-1602).
Antibody PAbl620 (Ab-5, Oncogene Science) reacts specifically with
wild type p53 (Ball, R. K. et al. (1984) EMBO J 3, 1485-1491).
MIgS-Kp I, a mouse monoclonal antibody of the same subclass as the
anti-p90 and anti-p53 antibodies, was used as a negative control at
similar working dilutions.
[0095] The avidin-biotin peroxidase method was performed on 5 um
thick frozen tissue sections fixed with cold methanol-acetone (1:1
dilution). Briefly, sections were incubated for 15 minutes with 10%
normal horse serum (Organon Tecknika Corp., Westchester, Pa.),
followed by a two hour incubation with appropriately diluted
primary antibodies (2A9, 4B2 and 4B11 were used at 1:100 dilution,
while 2A10 was used at 1:1000 dilution) (Ab-2 was used at 200
ng/ml; Ab-3 at 250 ng/ml and Ab-5 at 3 ug/ml). After extensive
washing, sections were subsequently incubated for 30 minutes with
biotinylated horse anti-mouse IgG antibodies at 1:200 dilution
(Vector Laboratories, Burlingame, Calif.) and avidin-biotin
peroxidase complexes (Vector Laboratories at 1:25 dilution for 30
minutes). Diaminobenzidine (0.06% DAB) was used as the final
chromogen and hematoxylin as the nuclear counterstain.
[0096] Immunohistochemical evaluation was done by at least two
independent investigators, scoring the estimated percentage of
tumor cells that showed nuclear staining. Both p90 and p53 nuclear
immunoreactivities were classified into three categories defined as
follows: negative (<20% tumor cells displaying nuclear
staining), heterogeneous (20-79% tumors cells with nuclear
reactivities), and homogeneous (.gtoreq.80% tumor cells with
intense nuclear staining).
[0097] Southern Blotting and Rflp Analyses.
[0098] A human dm2 cDNA fragment probe of 1.6 kb, pHDM (EcoRI), was
used in Southern blots to assess gene amplification. A b-actin
probe, (EcoRI), was used as a control. Two probes were used for the
analysis of allelic deletions of the short arm of chromosome 17,
PYNZ22 (17p13.3, D17S5, TaqI) and php53B (17p13.1, p53, BglII).
Southern analysis was performed as described (Presti, J. C. et al.
(1991) Cancer Res 51, 5405; Dalbagni, G. et al. (1993) Diagnostic
Molecular Pathology 2, 4-13). Briefly, DNA was extracted by the
non-organic method developed by Oncor (Oncor, Gaithersburg, Md.)
from paired normal and tumor samples, digested with the appropriate
restriction enzymes, electrophoresed in 0.7% agarose gel, and
blotted onto nylon membranes. The membranes were prehybridized with
Hybrisol I (Oncor) at 42.degree. C. for one hour, and hybridized
with probes labelled to high specific activity with [P32] dCTP
overnight. Membranes were then washed and subjected to
autoradiography using intensifying screens at -70.degree. C.
Densitometry using an Ultrascan XL Laser Densitometer (Pharmacia
LKB Biotechnology, Piscataway, N.J.), as well as a Betascope 630
Blot Analyzer (Betagen, Waltham, Mass.), was performed to confirm
the results. A case was considered to have a dm2 amplification when
it had at least 5 copies gene/cell. Loss of heterozygosity (LOH)
was defined as a greater than 40% decrease in signal intensity of
an allele in the tumor samples (Presti, J. C. et al. (1991) Cancer
Res 51, 5405; Olumi, A. F. et al. (1990) Cancer Res 50,
7081-7083).
[0099] Single Stranded Conformational Polymorphism (SSCP) Analysis
and DNA Sequencing.
[0100] These studies were performed according to a slight
modification of the method reported by Orita et al (Orita, M. et
al. (1989) Genomics 5, 874-879). Amplifications were performed
using 100 ng of genomic DNA extracted from the samples described
above. The primers used were obtained from intronic sequences
flanking exons 5 through 9 of the human p53 gene, sequences being
previously published (Moll, U. M. et al. (1992) Proc Natl Acad Sci
USA 89, 7262-7266). DNA was amplified following 30 cycles of PCR
(30s at 94.degree. C., 30s at 58.degree. C. for exons 8 and 9 and
63.degree. C. for exons 5, 6 and 7, and finally 60s for all samples
at 72.degree. C.) using a Thermal Cycler (Perkin Elmer Cetus).
Amplified samples were then denatured and loaded onto a
non-denaturing acrylamide gel containing 10% glycerol and run at
room temperature for 12-16 hours at 10-12 watts. Gels were dried at
80.degree. C. under vacuum and exposed to X-ray film at -70.degree.
C. for 4-16 hours.
[0101] Amplification of genomic DNA for sequencing assays was
independent of that used for SSCP analysis, using 35 cycles (60s at
94.degree. C., 60s at 58.degree. C. and 63.degree. C.--as above,
and 90s at 72.degree. C.). DNA fragments were isolated from 2% low
melting point agarose gels, purified and sequenced by the dideoxy
method (Sanger, F. et al. (1977) Proc Natl Acad Sci USA 74,
5463-5467). Both strands were sequenced for each DNA analyzed, and
genomic DNA from control samples containing wild-type p53 were
sequenced in parallel to confirm the mutations.
[0102] dm2 Amplification and Over-Production of dm2 Proteins.
[0103] Amplification of the dm2 gene was detected in 11 of 73 adult
soft tissue sarcomas (STS), ranging from 5- to 35-fold. Dm2
amplifications were more frequently detected in high grade (7
cases) than in low grade (4 cases) STS. Amplifications were more
commonly observed in metastatic (3 of 11 cases, 27%) than in
primary sarcomas (4 of 48 cases, 8%).
[0104] The pattern of immunostaining of anti-dm2 antibodies was
first assessed using 3T3-Balbc and 3T3-DM cells. A strong nuclear
staining was seen in DM cells, reported to have an amplified dm2
gene and to overexpress dm2 proteins; while Balb-c cells were
unreactive (FIG. 4) and have very low levels of dm2 proteins. Six
of the 11 amplified cases showed over 20% tumor cells displaying
nuclear immunoreactivities with anti-dm2 antibodies. However, the
remaining 5 cases were unreactive. Seventeen of the 62 cases with
an apparent non-amplified dm2 gene showed elevated levels of dm2
proteins as detected by the dm2 antibodies using tissue sections
(dm2-positive phenotype).
[0105] p53 Deletions, Point Mutations, and p53 Nuclear
Immunoreactivities.
[0106] 73 pairs of somatic and tumor DNA were examined with two
different probes for the short arm of chromosome 17. Deletions of
the short arm of chromosome 17 were found in 27 of 51 (53%)
informative cases examined. Loss of heterozygosity (LOH) of
chromosome 17p was observed in both low and high grade sarcomas.
Chromosome 17p LOH was more frequently found in metastatic (6 of 8
cases, 75%) than in primary (13 of 33 cases, 41%) tumors.
[0107] To further characterize the specific intragenic mutations of
p53 as they may relate to p53 overexpression in these cells, 73 STS
were analyzed using SSCP (exons 5 through 9) and those positive for
that assay were followed by DNA sequencing. Confirmation of the
presence of a mutation was revealed in 14 cases. Eleven of these 14
STS displayed p53 nuclear immunoreactivities for antibody PAb1801.
Point mutations were characterized by sequencing in 7 of these 11
sarcomas, 5 showed AT to GC transitions, while 2 were GC to AT
transitions. In 4 cases shifts in mobility were detected by SSCP,
but no sequencing was conducted to identify the mutation. Three of
the 14 mutant cases showed negative immunostaining results for
PAb1801. One mutation was identified in codon 165, producing a stop
codon. Another case had a C deletion at codon 278, producing a stop
codon at position 344. The other mutation occurred in exon 5
affecting a splice donor site. All informative mutants for
chromosome 17p status but one had a concomitant deletion of the
short arm. In addition, 13 cases showed a positive nuclear staining
signal without evidence of point mutations for the exons under
study.
[0108] Overall, 56 of the 211 STS analyzed displayed a positive
nuclear pattern of immunostaining for PAb1801 (Table 1). There was
a significant difference between p53-positive phenotype and tumor
grade. Moreover, patients affected with STS that showed p53 nuclear
immunoreactivities in over 20% tumor cells had significantly
reduced survival rates.
1TABLE 1 Soft Tissue Sarcomas High Expression of p53 and mdm-2 p53
over- expression p53 normal totals mdm-2 over- 22 54 76/211
expression (36%) mdm-2 normal 34 101 135/211 Totals 56/211 155/211
(26.5%)
[0109] Altered Genotype and Phenotype of dm2 and p53:
Clinicopathological Implications.
[0110] Only one of the subgroup of 73 cases exhibited an amplified
dm2 and a mutant p53 gene, and this was a metastatic fibrosarcoma.
However, 22 of a total cohort of 211 cases showed positive nuclear
immunoreactivities for both dm2 and p53 proteins in consecutive
tissue sections (Table 1). The pattern of staining of these
molecules, in cases on which they were co-expressed, was in general
heterogeneous. When comparing the combined phenotypes (Group A:
dm2-/p53-; Group B: dm2+/p53- and dm2-/p53+; and Group C:
dm2+/p53+) versus clinicopathological parameters, a correlation was
observed between the positive phenotype and variables for a poor
prognosis. The data support the conclusion that Group A correlates
with the best prognosis, and Group C correlates with the poorest
prognosis (see FIG. 3).
Example 3
[0111] p90 may be used to prepare antibodies that are capable of
immunoprecipitating p90 or co-immunoprecipitating p90 with p53. p90
may be isolated from the precipitate and purified. The anti-p90
antibodies may be polyclonal or monoclonal.
[0112] One method for preparing polyclonal antibodies to p90 is as
follows:
[0113] Procedure for p90 Antibody Production in Rabbits:
[0114] The p90 protein fragment is at a concentration of 0.7 mg/ml.
Immunizations are given as follows:
2 Day 0 25 .mu.g in 200 .mu.l PBS plus 200 .mu.l RIBI (adjuvant)
Day 7 50 .mu.g in 200 .mu.l PBS plus 200 .mu.l RIBI (adjuvant) Day
14 50 .mu.g in 200 .mu.l PBS plus 200 .mu.l RIBI (adjuvant) Day 21
REST Day 28 50 .mu.g in 200 .mu.l PBS plus 200 .mu.l RIBI Day 39
Bleed -- assay Day 52 Boost animal with 50 .mu.g in 200 .mu.l PBS
plus 200 .mu.l RIBI Day 59 Bleed --- 7 days after last injection
assay Day 62 Exsanguinate
[0115] Bleeds were assayed by ELISA, coating wells at 200 ng/well
overnight at 4.degree. C. Block 2% BSA for 1 hr. at 37.degree. C.
Make serum dilutions in 1% BSA and incubate 2 hrs. at 37.degree. C.
Secondary antibody dilutions (TAGO Goat anti-rabbit
peroxidase--cat.# 6430) in 1% BSA and incubate 37.degree. C. for 1
hr. Sera titered out to 1:25000 (cutoff absorbance 0.400 at 450 nm)
for the first assay and >50000 for the final assay. Develop with
Kirkegaard & Perry TMB Peroxidase Substrate Solution(s) and
read absorbance at 450 nm.
Example 4
[0116] The hybridomas 1F5, 6C10, 1D6, 4B2, 2E12, 3F3, 3G5, 3F8,
6H7, 2A9, 3G9, 1D11, 2A10, 1G2, 4B11 and 5B10 deposited with the
ATCC (see above) produce monoclonal antibodies against p90 protein
epitopes (see FIG. 1). The following protocols use these monoclonal
antibodies and other anti-p90 antibodies as primary antibodies for
detecting the DM2 gene-coded product, the p90 protein, by
immunohistochemical methods in tissues, preferably human
tissues.
[0117] The avidin-biotin immunoperoxidase technique is suitable due
to the high sensitivity that it renders. Sections of human normal
and tumor tissues are cut using a cryostat and placed on
microslides. These sections are then incubated with blocking serum,
followed by hydrogen peroxide and avidin-biotin blocking. Primary
antibodies, i.e. anti-p90 antibodies, are then used at an
appropriate concentration. The appropriate concentration is
empirically determined for each antibody by performing titrations.
Sections are then incubated with biotinylated secondary horse
anti-mouse antibodies, followed by avidin-biotin peroxidase
complexes. The final reaction is developed using diaminobenzidine.
Sections are then counterstained with hematoxylin and mounted with
permount for final analysis.
[0118] Using such imnunohistochemical methods, typically only
elevated levels of the p90 protein, not normal levels, are
detectable in the tissues since, for those tissues examined in
these cases, the monoclonal antibodies can detect only elevated
levels of p90 protein in cells.
Immunohistochemistry Avidin-Biotin-Peroxidase Method Paraffin
Embedded Tissue Sections
[0119] 1) Place 5 .mu.m tissue sections on poly-L-lysine coated
slides.
[0120] 2) Place sections in 60.degree. C. oven for 30 minutes to
melt paraffin.
[0121] 3) Cool slides at room temperature then process for
deparaffinization and rehydration:
[0122] xylene--3 times (5 minutes each)
[0123] 100% ethanol--3 times (3 minutes each)
[0124] 95% ethanol--3 times (3 minutes each)
[0125] 4) Wash in distilled H.sub.2O and transfer to PBS.
[0126] 5) Quench with 1% H.sub.2O.sub.2 for 15 minutes to eliminate
endogenous peroxidase activity.
[0127] 6) Wash in PBS--3 times.
[0128] 7) In tissues that contain biotin (such as liver, kidney,
brain, etc.), apply Avidin-Biotin blocking kit (Vector) in order to
eliminate endogenous biotin activity. Solutions should be applied
subsequently (Avidin, then biotin) and slides should be incubated
for 15 minutes with each solution.
[0129] 8) Wash in PBS.
[0130] 9) Enzyme digestion--enzyme selection determined empirically
as optimally for each antibody.
[0131] Common Enzymes:
[0132] Pepsin (Porcine Stomach Mucosa, Sigma): HCL (250 ml
distilled H.sub.2O+200 .mu.l HCl )+0.25 grams
pepsin.fwdarw.incubation 30 minutes.
[0133] Trypsin (Bovine Pancreas Type I, Sigma): 250 ml TRIS+0.0625
grams trypsin.fwdarw.incubate for 5 minutes; wash in distilled
H.sub.2O, then incubate for 15 minutes with trypsin inhibitor
(Sigma): 250 ml PBS+0.025 gms trypsin inhibitor.
[0134] Pronase (Calbiochem Behring): 250 ml TRIS+0.0055 gms
pronase.fwdarw.incubate for 4 minutes;
[0135] Ficin (suspension--Sigma)--ready to use, dropwise,
incubation 45 minutes.
[0136] Saponin (detergent--Sigma): 250 ml distilled H.sub.2O+0.125
gms Saponin.fwdarw.incubation 30 minutes.
[0137] 10) Wash slides in distilled H.sub.2O and transfer to
PBS.
[0138] 11) Apply blocking serum--10% normal serum (species
specific--same species as the secondary antibody)--incubation 20-30
minutes.
[0139] 12) Vacuum suction off the blocking serum and apply
appropriately diluted primary antibody--incubation overnight at
4.degree. C. in a humid chamber. The primary antibody is selected
from the anti-p90 monoclonal antibodies produced by the following
hybridomas: 1FS, 6C10, 1D6, 4B2, 2E12, 3F3, 3G5, 3F8, 6H7, 2A9,
3G9, 1D11, 2A10, 1G2, 4B11 and 5B10. The appropriate dilution of
the primary antibody is an empirically determined optimal
concentration.
[0140] 13) Extensive washing with PBS.fwdarw.3 changes (5 minutes
each)
[0141] 14) Apply appropriately diluted biotinylated secondary
antibody.fwdarw.30 minutes incubation.
[0142] 15) Wash with PBS (3.times.5 minutes).
[0143] 16) Avidin-biotin complex (Vector) diluted 1:25 (equal ratio
A B).fwdarw.incubation 30 minutes.
[0144] 17) Wash with PBS (3.times.5 minutes).
[0145] 18) Substrate chromogen solution:
[0146] peroxidase-diaminobenzadine (0.06% DAB)
[0147] (5 mg DAB/ 100 ml PBS+100 ul 0.3% H.sub.2O.sub.2)
[0148] Incubate until desired color intensity has developed
(approximately 5 minutes)
[0149] 19) Hematoxylin counterstain.
Immunohistochemistry Avidin-Biotin-Peroxidase Method--Frozen Tissue
Sections
[0150] 1. 5 .mu.m frozen tissue sections--leave at room temperature
for at least 30 minutes to thaw and dry sections.
[0151] 2. Apply proper fixative for 10 minutes (fixative optimally
chosen for each antibody).
[0152] 3. Quench with 0.1% H.sub.2O.sub.2 for 15 minutes to
eliminate endogenous peroxidase activity.
[0153] 4. Wash in PBS--3.times..
[0154] 5. Avidin-biotin blocking kit in tissues rich with
endogenous biotin. Avidin--incubate 15 minutes, wash with PBS and
then biotin--15 minutes.
[0155] 6. Wash in PBS--3.times..
[0156] 7. Apply blocking serum--10% normal serum (species specific,
same species as the secondary antibody) 10-30 minutes incubation in
humid chamber.
[0157] 8. Suction off the blocking serum and apply appropriately
diluted primary antibody incubation 1-2 hours. The primary antibody
is selected from the anti-p90 monoclonal antibodies produced by the
following hybridomas: 1F5, 6C10, 1D6, 4B2, 2E12, 3F3, 3G5, 3F8,
6H7, 2A9, 3G9, 1D11, 2A10, 1G2, 4B11 and 5B10. The appropriate
dilution of the primary antibody is an empirically determined
optimal concentration, typically 1-1000 volume/volume of hybridoma
supernatant/phosphate buffered saline (PBS).
[0158] 9. Wash extensively in PBS.
[0159] 10. Apply appropriately diluted biotinylated secondary
antibody--30 minutes incubation.
[0160] 11. Wash with PBS--3.times..
[0161] 12. Avidin-biotin complex (Vector) dilution 1:25 (equal
ratio A:B)--30 minutes.
[0162] 13. Wash PBS and PBS/Triton--3.times..
[0163] 14. Substrate chromagen solution peroxidase-diaminobenzidine
(0.06% DAB) approximately 5 minutes.
[0164] 15. Hematoxylin counterstain.
* * * * *