U.S. patent application number 13/757610 was filed with the patent office on 2013-08-01 for polymorphisms for predicting disease and treatment outcome.
The applicant listed for this patent is Heinz-Josef Lenz, David Jong-Han Park, Jan Stoehlmacher. Invention is credited to Heinz-Josef Lenz, David Jong-Han Park, Jan Stoehlmacher.
Application Number | 20130196955 13/757610 |
Document ID | / |
Family ID | 31192379 |
Filed Date | 2013-08-01 |
United States Patent
Application |
20130196955 |
Kind Code |
A1 |
Lenz; Heinz-Josef ; et
al. |
August 1, 2013 |
POLYMORPHISMS FOR PREDICTING DISEASE AND TREATMENT OUTCOME
Abstract
The invention provides compositions and methods for determining
the increased risk for recurrence of certain cancers and the
likelihood of successful treatment with one or both of chemotherapy
and radiation therapy. The methods comprising determining the type
of genomic polymorphism present in a predetermined region of the
gene of interest isolated from the subject or patient. Also
provided are nucleic acid probes and kits for determining a
patient's cancer risk and treatment response.
Inventors: |
Lenz; Heinz-Josef; (Los
Angeles, CA) ; Stoehlmacher; Jan; (Dresden, DE)
; Park; David Jong-Han; (Rowland Heights, CA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Lenz; Heinz-Josef
Stoehlmacher; Jan
Park; David Jong-Han |
Los Angeles
Dresden
Rowland Heights |
CA
CA |
US
DE
US |
|
|
Family ID: |
31192379 |
Appl. No.: |
13/757610 |
Filed: |
February 1, 2013 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
13275225 |
Oct 17, 2011 |
|
|
|
13757610 |
|
|
|
|
12650323 |
Dec 30, 2009 |
|
|
|
13275225 |
|
|
|
|
10522664 |
Aug 3, 2005 |
7662553 |
|
|
PCT/US03/24065 |
Jul 31, 2003 |
|
|
|
12650323 |
|
|
|
|
60400276 |
Jul 31, 2002 |
|
|
|
60400253 |
Jul 31, 2002 |
|
|
|
60400250 |
Jul 31, 2002 |
|
|
|
60400249 |
Jul 31, 2002 |
|
|
|
Current U.S.
Class: |
514/158 ;
435/6.11 |
Current CPC
Class: |
C12Q 2600/106 20130101;
C12Q 1/6886 20130101; A61P 35/04 20180101; A61P 43/00 20180101;
A61K 31/415 20130101; C12Q 2600/118 20130101; A61P 35/00 20180101;
A61K 31/635 20130101 |
Class at
Publication: |
514/158 ;
435/6.11 |
International
Class: |
C12Q 1/68 20060101
C12Q001/68; A61K 31/635 20060101 A61K031/635 |
Claims
1. A method for selecting a therapeutic regimen for treating a
cancer in a patient wherein a chemotherapeutic drug is administered
to the patient, the method comprising screening a suitable cell or
tissue sample isolated from said patient for a genomic polymorphism
or genotype that is correlative to treatment outcome of the
cancer.
2. The method of claim 1, wherein the cancer is a cancer that can
be treated by the administration of a chemotherapeutic drug
selected from the group consisting of fluoropyrimidine or a
platinum drug.
3. The method of claim 1, wherein the cancer is selected from the
group consisting of esophageal cancer, gastric cancer, colon
cancer, rectal cancer, colorectal cancer and lung cancer.
4. The method of claim 2, wherein the cancer treatment further
comprises radiation therapy.
5. The method of claim 1, wherein said genomic polymorphism occurs
in the gene selected from the group consisting of thymidylate
synthase gene, excision repair complementation group gene (ERCC1),
VEGF, ERC2 gene, XRCC-1 gene, human glutathione s-transferase P1
gene, epidermal growth factor receptor gene, matrix
metalloproteinase genes (-1, and -3), interleukin 8 (IL-8) gene,
D-pyrimidine dehydrogenase (DPD) and CXC chemokine.
6. The method of claim 5, wherein the genotype is high expression
of a gene selected from the group consisting of thymidylate
synthase, D-pyrimidine dehydrogenase (DPD) ERCC1 and VEGF and said
tissue sample is normal tissue that corresponds to the tumor
type.
7. A method for reducing chemically induced neurotoxicity
associated with cancer chemotherapy in a patient comprising
administering to said subject an effective amount of a COX-2
inhibitor to a patient in need thereof.
8. The method of claim 7, wherein the chemotherapy comprises
administration of oxaliplatin.
9. The method of claim 7, wherein the chemotherapy comprises
administration of 5-FU.
10. A method for determining if a human patient is more likely to
experience tumor recurrence after surgical removal of said tumor,
comprising determining the expression level of a gene selected from
the group consisting of TS, DPD, ERCC1 and VEGF, in a cell or
sample isolated from normal tissue adjacent to said tumor and
correlating said expression level with normal levels, wherein
overexpression of said gene is predictive to identify patients at
risk for tumor recurrence.
11. The method of claim 11, wherein the tumor is associated with
rectal cancer.
Description
CROSS-REFERENCE TO RELATED PATENT APPLICATIONS
[0001] This application is a continuation under 35 U.S.C. .sctn.120
of U.S. patent application Ser. No. 13/275,225, filed Oct. 17,
2011, which is a continuation of U.S. application Ser. No.
12/650,323, filed Dec. 30, 2009, which is a continuation of U.S.
application Ser. No. 10/522,664, filed Aug. 3, 2005, now U.S. Pat.
No. 7,662,553, issued Feb. 16, 2010, which is a national stage
application under 35 U.S.C. .sctn.371 of International Application
No. PCT/US2003/024065, filed Jul. 31, 2003, which in turn claims
priority under 35 U.S.C. .sctn.119(e) to U.S. Provisional
Application Nos. 60/400,249; 60/400,250; 60/400,253; and
60/400,276, each filed on the same day of Jul. 31, 2002. The
contents of these applications are hereby incorporated by reference
into the present disclosure.
FIELD OF THE INVENTION
[0002] This invention relates to the field of pharmacogenomics and
specifically to the application of genetic polymorphism to
diagnosing and treating diseases.
BACKGROUND OF THE INVENTION
[0003] In nature, organisms of the same species usually differ from
each other in some aspects, e.g., their appearance. The differences
are genetically determined and are referred to as polymorphism. At
many gene loci, two or more alleles may occur (genetic
polymorphism). Genetic polymorphism is defined as the occurrence in
a population of two or more genetically determined alternative
phenotypes due to different alleles. Polymorphism can be observed
at the level of the whole individual (phenotype), in variant forms
of proteins and blood group substances (biochemical polymorphism),
morphological features of chromosomes (chromosomal polymorphism) or
at the level of DNA in differences of nucleotides (DNA
polymorphism).
[0004] Polymorphism may play a role in determining individual
differences in the response to drugs. Cancer chemotherapy is
limited by the predisposition of specific populations to drug
toxicity or poor drug response. Thus, for example, pharmacogenetics
(the effect of genetic differences on drug response) has been
applied in cancer chemotherapy to understand the significant
inter-individual variations in responses and toxicities to the
administration of anti-cancer drugs, which may be due to genetic
alterations in drug metabolizing enzymes or receptor expression.
See co-pending U.S. application Ser. No. 09/715,764, incorporated
by reference herein.
[0005] Polymorphism is also associated with cancer susceptibility
(oncogenes, tumor suppressor genes and genes of enzymes involved in
metabolic pathways) of individuals. In patients younger than 35
years, several markers of increased cancer risk have been
identified. For example, prostate specific antigen (PSA) can be
used for the early detection of prostate cancer in asymptomatic
younger males, while particular cytochrome P4501A1 and gluthathione
S-transferase M1 genotypes influence the risk of developing
prostate cancer in younger patients. Similarly, mutations in the
tumor suppressor gene, p53, are associated with brain tumors in
young adults.
[0006] Thus a need exists to identify genetic markers that are
predictive of drug toxicity or poor drug response. This invention
satisfies this need and provides related advantages as well.
DESCRIPTION OF THE EMBODIMENTS
[0007] In one embodiment, the invention comprises the use of the
allelic variant of the polymorphic region of the gene of interest
to select a cancer treatment protocol. These methods of use include
prognostic, diagnostic, and therapeutic methods. In one aspect, the
variant of interest is expressed as a gene that is highly expressed
(based on mRNA expression levels) of the gene in the adjacent and
corresponding "normal" tissue remaining after surgical resection.
In another aspect, the variant of interest is detected at the
genomic level and can comprise regions of the gene which are, or
are not, ultimately transcribed and translated into protein. For
example, such regions include, but are not limited to the
untranslated promoter regions or the untranslated 3' region of the
gene.
[0008] Methods to detect polymorphisms include using nucleic acids
encompassing the polymorphic region as probes or primers to
determine whether a subject has or is at risk of developing cancer
and/or the subject's response to chemotherapy. Alternatively, mRNA
levels can be detected using nucleic acid probes or arrays.
[0009] In one aspect, the cancer comprises a cancer or neoplasm
that is "treatable" by use of platinum therapy, e.g., oxaliplatin
or cisplatin or 5-fluorouridine (5-FU) and the orally available FU
therapy, sold under the name Xeloda (Roche). Non-limiting examples
of such cancers include, but are not limited to rectal cancer,
colorectal cancer, colon cancer, gastric cancer, lung cancer,
esophageal cancers. In one aspect, the sample to be tested is the
actual tumor tissue. In another aspect, the sample to be tested in
the method is normal "corresponding" tissue to the tumor tissue,
e.g., normal lung tissue is considered to be the corresponding
normal tissue to lung cancer tissue. In yet a further example, the
sample is any tissue of the patient, and can include peripheral
blood lymphocytes.
[0010] In another embodiment, the invention provides a kit for
amplifying and/or for determining the molecular structure of at
least a portion of the gene of interest, comprising a probe or
primer capable of hybridizing to the gene of interest and
instructions for use. In one embodiment, the probe or primer is
capable of hybridizing to an allelic variant of the gene of
interest.
[0011] Other features and advantages of the invention will be
apparent from the following detailed description and claims.
BRIEF DESCRIPTION OF THE FIGURES
[0012] FIG. 1 shows time to local recurrence in rectal cancer based
on molecular parameters.
[0013] FIGS. 2A through 2D show the estimated probability of
recurrence free survival in patients with rectal cancer versus gene
expressions of TS, DPD, ERCC1, and VEGF in normal rectal tissue.
See Experimental Number 1 for experimental details.
[0014] FIG. 3 shows the estimated probability of recurrence free
survival in patients with rectal cancer versus gene expression of
VEGF in rectal cancer tissue. See Experimental Number 1 for
experimental details.
MODES FOR CARRYING OUT THE INVENTION
[0015] The present invention provides methods and kits to determine
a subject risk of cancer and response to cancer treatment by
determining the subject's genotype at the gene of interest. Other
aspects of the invention are described below or will be apparent to
one of skill in the art in light of the present disclosure.
[0016] Throughout this disclosure, various publications, patents
and published patent specifications are referenced by an
identifying citation. The disclosures of these publications,
patents and published patent specifications are hereby incorporated
by reference into the present disclosure to more fully describe the
state of the art to which this invention pertains.
[0017] The practice of the present invention employs, unless
otherwise indicated, conventional techniques of molecular biology
(including recombinant techniques), microbiology, cell biology,
biochemistry and immunology, which are within the skill of the art.
Such techniques are explained fully in the literature for example
in the following publications. See, e.g., Sambrook et al. MOLECULAR
CLONING: A LABORATORY MANUAL, 2.sup.nd edition (1989); CURRENT
PROTOCOLS IN MOLECULAR BIOLOGY (F. M. Ausubel et al. eds. (1987));
the series METHODS IN ENZYMOLOGY (Academic Press, Inc., N.Y.); PCR:
A PRACTICAL APPROACH (M. MacPherson et al. IRL Press at Oxford
University Press (1991)); PCR 2: A PRACTICAL APPROACH (M. J.
MacPherson, B. D. Hames and G. R. Taylor eds. (1995)); ANTIBODIES,
A LABORATORY MANUAL (Harlow and Lane eds. (1988)); ANIMAL CELL
CULTURE (R. L. Freshney ed. (1987)); OLIGONUCLEOTIDE SYNTHESIS (M.
J. Gait ed. (1984)); Mullis et al. U.S. Pat. No. 4,683,195; NUCLEIC
ACID HYBRIDIZATION (B. D. Hames & S. J. Higgins eds. (1984));
TRANSCRIPTION AND TRANSLATION (B. D. Hames & S. J. Higgins eds.
(1984)); IMMOBILIZED CELLS AND ENZYMES (IRL Press (1986)); B.
Perbal, A PRACTICAL GUIDE TO MOLECULAR CLONING (1984); GENE
TRANSFER VECTORS FOR MAMMALIAN CELLS (J. H. Miller and M. P. Calos
eds. (1987) Cold Spring Harbor Laboratory); IMMUNOCHEMICAL METHODS
IN CELL AND MOLECULAR BIOLOGY (Mayer and Walker, eds., Academic
Press, London (1987)); HANDBOOK OF EXPERIMENTAL IMMUNOLOGY, Volumes
I-IV (D. M. Weir and C. C. Blackwell, eds. (1986)); MANIPULATING
THE MOUSE EMBRYO (Cold Spring Harbor Laboratory Press, Cold Spring
Harbor, N.Y. (1986)).
DEFINITIONS
[0018] The term "allele", which is used interchangeably herein with
"allelic variant" refers to alternative forms of a gene or portions
thereof. Alleles occupy the same locus or position on homologous
chromosomes. When a subject has two identical alleles of a gene,
the subject is said to be homozygous for the gene or allele. When a
subject has two different alleles of a gene, the subject is said to
be heterozygous for the gene. Alleles of a specific gene can differ
from each other in a single nucleotide, or several nucleotides, and
can include substitutions, deletions, and insertions of
nucleotides. An allele of a gene can also be a form of a gene
containing a mutation.
[0019] The term "allelic variant of a polymorphic region of the
gene of interest" refers to a region of the gene of interest having
one of a plurality of nucleotide sequences found in that region of
the gene in other individuals.
[0020] "Cells," "host cells" or "recombinant host cells" are terms
used interchangeably herein. It is understood that such terms refer
not only to the particular subject cell but to the progeny or
potential progeny of such a cell. Because certain modifications may
occur in succeeding generations due to either mutation or
environmental influences, such progeny may not, in fact, be
identical to the parent cell, but are still included within the
scope of the term as used herein.
[0021] The expression "amplification of polynucleotides" includes
methods such as PCR, ligation amplification (or ligase chain
reaction, LCR) and amplification methods based on the use of Q-beta
replicase. These methods are well known and widely practiced in the
art. See, e.g., U.S. Pat. Nos. 4,683,195 and 4,683,202 and Innis et
al., 1990 (for PCR); and Wu, D. Y. et al. (1989) Genomics 4:560-569
(for LCR). In general, the PCR procedure describes a method of gene
amplification which is comprised of (i) sequence-specific
hybridization of primers to specific genes within a DNA sample (or
library), (ii) subsequent amplification involving multiple rounds
of annealing, elongation, and denaturation using a DNA polymerase,
and (iii) screening the PCR products for a band of the correct
size. The primers used are oligonucleotides of sufficient length
and appropriate sequence to provide initiation of polymerization,
i.e. each primer is specifically designed to be complementary to
each strand of the genomic locus to be amplified.
[0022] Reagents and hardware for conducting PCR are commercially
available. Primers useful to amplify sequences from a particular
gene region are preferably complementary to, and hybridize
specifically to sequences in the target region or in its flanking
regions. Nucleic acid sequences generated by amplification may be
sequenced directly. Alternatively the amplified sequence(s) may be
cloned prior to sequence analysis. A method for the direct cloning
and sequence analysis of enzymatically amplified genomic segments
is known in the art.
[0023] The term "encode" as it is applied to polynucleotides refers
to a polynucleotide which is said to "encode" a polypeptide if, in
its native state or when manipulated by methods well known to those
skilled in the art, it can be transcribed and/or translated to
produce the mRNA for the polypeptide and/or a fragment thereof. The
antisense strand is the complement of such a nucleic acid, and the
encoding sequence can be deduced therefrom.
[0024] The term "genotype" refers to the specific allelic
composition of an entire cell or a certain gene, whereas the term
"phenotype` refers to the detectable outward manifestations of a
specific genotype.
[0025] As used herein, the term "gene" or "recombinant gene" refers
to a nucleic acid molecule comprising an open reading frame and
including at least one exon and (optionally) an intron sequence.
The term "intron" refers to a DNA sequence present in a given gene
which is spliced out during mRNA maturation.
[0026] "Homology" or "identity" or "similarity" refers to sequence
similarity between two peptides or between two nucleic acid
molecules. Homology can be determined by comparing a position in
each sequence which may be aligned for purposes of comparison. When
a position in the compared sequence is occupied by the same base or
amino acid, then the molecules are homologous at that position. A
degree of homology between sequences is a function of the number of
matching or homologous positions shared by the sequences. An
"unrelated" or "non-homologous" sequence shares less than 40%
identity, though preferably less than 25% identity, with one of the
sequences of the present invention.
[0027] The term "a homolog of a nucleic acid" refers to a nucleic
acid having a nucleotide sequence having a certain degree of
homology with the nucleotide sequence of the nucleic acid or
complement thereof. A homolog of a double stranded nucleic acid is
intended to include nucleic acids having a nucleotide sequence
which has a certain degree of homology with or with the complement
thereof. In one aspect, homologs of nucleic acids are capable of
hybridizing to the nucleic acid or complement thereof.
[0028] The term "interact" as used herein is meant to include
detectable interactions between molecules, such as can be detected
using, for example, a hybridization assay. The term interact is
also meant to include "binding" interactions between molecules.
Interactions may be, for example, protein-protein, protein-nucleic
acid, protein-small molecule or small molecule-nucleic acid in
nature.
[0029] The term "isolated" as used herein with respect to nucleic
acids, such as DNA or RNA, refers to molecules separated from other
DNAs or RNAs, respectively, that are present in the natural source
of the macromolecule. The term isolated as used herein also refers
to a nucleic acid or peptide that is substantially free of cellular
material, viral material, or culture medium when produced by
recombinant DNA techniques, or chemical precursors or other
chemicals when chemically synthesized. Moreover, an "isolated
nucleic acid" is meant to include nucleic acid fragments which are
not naturally occurring as fragments and would not be found in the
natural state. The term "isolated" is also used herein to refer to
polypeptides which are isolated from other cellular proteins and is
meant to encompass both purified and recombinant polypeptides.
[0030] The term "mismatches" refers to hybridized nucleic acid
duplexes which are not 100% homologous. The lack of total homology
may be due to deletions, insertions, inversions, substitutions or
frameshift mutations.
[0031] As used herein, the term "nucleic acid" refers to
polynucleotides such as deoxyribonucleic acid (DNA), and, where
appropriate, ribonucleic acid (RNA). The term should also be
understood to include, as equivalents, derivatives, variants and
analogs of either RNA or DNA made from nucleotide analogs, and, as
applicable to the embodiment being described, single (sense or
antisense) and double-stranded polynucleotides.
Deoxyribonucleotides include deoxyadenosine, deoxycytidine,
deoxyguanosine, and deoxythymidine. For purposes of clarity, when
referring herein to a nucleotide of a nucleic acid, which can be
DNA or an RNA, the terms "adenosine", "cytidine", "guanosine", and
thymidine" are used. It is understood that if the nucleic acid is
RNA, a nucleotide having a uracil base is uridine.
[0032] The terms "oligonucleotide" or "polynucleotide", or
"portion," or "segment" thereof refer to a stretch of
polynucleotide residues which is long enough to use in PCR or
various hybridization procedures to identify or amplify identical
or related parts of mRNA or DNA molecules. The polynucleotide
compositions of this invention include RNA, cDNA, genomic DNA,
synthetic forms, and mixed polymers, both sense and antisense
strands, and may be chemically or biochemically modified or may
contain non-natural or derivatized nucleotide bases, as will be
readily appreciated by those skilled in the art. Such modifications
include, for example, labels, methylation, substitution of one or
more of the naturally occurring nucleotides with an analog,
internucleotide modifications such as uncharged linkages (e.g.,
methyl phosphonates, phosphotriesters, phosphoamidates, carbamates,
etc.), charged linkages (e.g., phosphorothioates,
phosphorodithioates, etc.), pendent moieties (e.g., polypeptides),
intercalators (e.g., acridine, psoralen, etc.), chelators,
alkylators, and modified linkages (e.g., alpha anomeric nucleic
acids, etc.). Also included are synthetic molecules that mimic
polynucleotides in their ability to bind to a designated sequence
via hydrogen bonding and other chemical interactions. Such
molecules are known in the art and include, for example, those in
which peptide linkages substitute for phosphate linkages in the
backbone of the molecule.
[0033] The term "nucleotide sequence complementary to the
nucleotide sequence set forth in SEQ ID NO: x" refers to the
nucleotide sequence of the complementary strand of a nucleic acid
strand having SEQ ID NO: x. The term "complementary strand" is used
herein interchangeably with the term "complement". The complement
of a nucleic acid strand can be the complement of a coding strand
or the complement of a non-coding strand. When referring to double
stranded nucleic acids, the complement of a nucleic acid having SEQ
ID NO: x refers to the complementary strand of the strand having
SEQ ID NO: x or to any nucleic acid having the nucleotide sequence
of the complementary strand of SEQ ID NO: x. When referring to a
single stranded nucleic acid having the nucleotide sequence SEQ ID
NO: x, the complement of this nucleic acid is a nucleic acid having
a nucleotide sequence which is complementary to that of SEQ ID NO:
x. The nucleotide sequences and complementary sequences thereof are
always given in the 5' to 3' direction. The term "complement" and
"reverse complement" are used interchangeably herein.
[0034] A "non-human animal" of the invention can include mammals
such as rodents, non-human primates, sheep, goats, horses, dogs,
cows, chickens, amphibians, reptiles, etc. Preferred non-human
animals are selected from the rodent family including rat and
mouse, most preferably mouse, though transgenic amphibians, such as
members of the Xenopus genus, and transgenic chickens can also
provide important tools for understanding and identifying agents
which can affect, for example, embryogenesis and tissue formation.
The term "chimeric animal" is used herein to refer to animals in
which an exogenous sequence is found, or in which an exogenous
sequence is expressed in some but not all cells of the animal. The
term "tissue-specific chimeric animal" indicates that an exogenous
sequence is present and/or expressed or disrupted in some tissues,
but not others.
[0035] The term "polymorphism" refers to the coexistence of more
than one form of a gene or portion thereof. A portion of a gene of
which there are at least two different forms, i.e., two different
nucleotide sequences, is referred to as a "polymorphic region of a
gene". A polymorphic region can be a single nucleotide, the
identity of which differs in different alleles.
[0036] A "polymorphic gene" refers to a gene having at least one
polymorphic region.
[0037] This invention provides a method for selecting a therapeutic
regimen or determining if a certain therapeutic regimen is more
likely to treat a cancer or is the appropriate chemotherapy for
that patient than other chemotherapies that may be available to the
patient. In general, a therapy is considered to "treat" cancer if
it provides one or more of the following treatment outcomes: reduce
or delay recurrence of the cancer after the initial therapy;
increase median survival time or decrease metastases. The method is
particularly suited to determining which patients will be
responsive or experience a positive treatment outcome to a
chemotherapeutic regimen involving administration of a
fluoropyrimidine drug such as 5-FU or a platinum drug such as
oxaliplatin or cisplatin. In one embodiment, the chemotherapeutic
regimen further comprises radiation therapy.
[0038] The method comprises isolating a suitable cell or tissue
sample from the patient and screening for a genomic polymorphism or
genotype that has been correlated by the Applicants to treatment
outcome of the cancer. In one aspect, the cancer is a cancer that
can be treated by the administration of a chemotherapeutic drug
selected from the group consisting of fluoropyrimidine or a
platinum drug. In another aspect, the cancer is selected from the
group consisting of esophageal cancer, gastric cancer, colon
cancer, rectal cancer, colorectal cancer and lung cancer.
[0039] In one aspect, the polymorphism is present in a "silent"
region of the gene, in another it is in the promoter region and in
yet another it is in the 3' untranslated region. In yet a further
embodiment, the polymorphism increases expression at the mRNA
level.
[0040] In one embodiment, the suitable tissue or cell sample
comprises normal tissue adjacent to the site of tumor biopsy or
resection. For example, one would select normal rectal tissue
adjacent to the site of rectal cancer tumor removal. As used
herein, "adjacent" mean about 0.5 mm, or about 1.0 mm, or about 1.5
mm, or about 2.0 mm or about 2.5 mm, or about 3.0 mm, or about 3.5
mm, or about 4.0 mm or about 4.5 mm or about 5.0 mm or
alternatively any distance the only limitation being that the
normal tissue be of the same type as the tumor or neoplasm.
[0041] In another embodiment, the tissue is the tumor tissue
itself. In yet a further embodiment, any cell expected to carry the
gene of interest, when the polymorphism is, for example, genetic,
such as a peripheral blood lymphocyte isolated from the patient, is
a suitable cell or tissue sample.
[0042] Genetic polymorphisms that can be predictive of outcome
include, but are not limited to polymorphisms occurring in a gene
selected from the group consisting of thymidylate synthase gene,
excision repair complementation group gene (ERCC1), VEGF, ERC2
gene, XRCC-1 gene, human glutathione s-transferase P1 gene,
epidermal growth factor receptor gene, matrix metalloproteinase
genes (-1, and -3), interleukin 8 (IL-8) gene, D-pyrimidine
dehydrogenase (DPD) and CXC chemokine.
[0043] This invention also provides a method for reducing
chemically induced neurotoxicity associated with cancer
chemotherapy in a patient comprising administering to the patient
an effective amount of a COX-2 inhibitor or its equivalent to a
patient in need thereof. In one embodiment, the neurotoxicity is
the result of administration of chemotherapy comprising
oxaliplatin, cisplatin or a fluoropyrimidine such as 5-FU or
Xeloda.
[0044] A method for determining if a human patient is more likely
to experience tumor recurrence after surgical removal of said
tumor, comprising determining the expression level of a gene
selected from the group consisting of TS, D-pyrimidine
dehydrogenase (DPD), ERCC1 and VEGF, in a cell or sample isolated
from normal tissue adjacent to said tumor and correlating said
expression level with normal levels, wherein overexpression of said
gene is predictive to identify patients at risk for tumor
recurrence. In one aspect, the tumor is associated with rectal
cancer.
[0045] The invention described herein relates to methods and
compositions for determining and identifying the allele present at
the gene of interest's locus. This information is useful to
diagnose and prognose disease progression as well as select the
most effective treatment among treatment options. Probes can be
used to directly determine the genotype of the sample or can be
used simultaneously with or subsequent to amplification. The term
"probes" includes naturally occurring or recombinant single- or
double-stranded nucleic acids or chemically synthesized nucleic
acids. They may be labeled by nick translation, Klenow fill-in
reaction, PCR or other methods known in the art. Probes of the
present invention, their preparation and/or labeling are described
in Sambrook et al. (1989) supra. A probe can be a polynucleotide of
any length suitable for selective hybridization to a nucleic acid
containing a polymorphic region of the invention. Length of the
probe used will depend, in part, on the nature of the assay used
and the hybridization conditions employed.
[0046] In one embodiment of the invention, probes are labeled with
two fluorescent dye molecules to form so-called "molecular beacons"
(Tyagi, S, and Kramer, F. R. (1996) Nat. Biotechnol. 14:303-8).
Such molecular beacons signal binding to a complementary nucleic
acid sequence through relief of intramolecular fluorescence
quenching between dyes bound to opposing ends on an oligonucleotide
probe. The use of molecular beacons for genotyping has been
described (Kostrikis, L. G. (1998) Science 279:1228-9) as has the
use of multiple beacons simultaneously (Marras, S. A. (1999) Genet.
Anal. 14:151-6). A quenching molecule is useful with a particular
fluorophore if it has sufficient spectral overlap to substantially
inhibit fluorescence of the fluorophore when the two are held
proximal to one another, such as in a molecular beacon, or when
attached to the ends of an oligonucleotide probe from about 1 to
about 25 nucleotides.
[0047] Labeled probes also can be used in conjunction with
amplification of a polymorphism. (Holland et al. (1991) Proc. Natl.
Acad. Sci. 88: 7276-7280). U.S. Pat. No. 5,210,015 by Gelfand et
al. describe fluorescence-based approaches to provide real time
measurements of amplification products during PCR. Such approaches
have either employed intercalating dyes (such as ethidium bromide)
to indicate the amount of double-stranded DNA present, or they have
employed probes containing fluorescence-quencher pairs (also
referred to as the "Taq-Man" approach) where the probe is cleaved
during amplification to release a fluorescent molecule whose
concentration is proportional to the amount of double-stranded DNA
present. During amplification, the probe is digested by the
nuclease activity of a polymerase when hybridized to the target
sequence to cause the fluorescent molecule to be separated from the
quencher molecule, thereby causing fluorescence from the reporter
molecule to appear. The Taq-Man approach uses a probe containing a
reporter molecule-quencher molecule pair that specifically anneals
to a region of a target polynucleotide containing the
poymorphism.
[0048] Probes can be affixed to surfaces for use as "gene chips."
Such gene chips can be used to detect genetic variations by a
number of techniques known to one of skill in the art. In one
technique, oligonucleotides are arrayed on a gene chip for
determining the DNA sequence of a by the sequencing by
hybridization approach, such as that outlined in U.S. Pat. Nos.
6,025,136 and 6,018,041. The probes of the invention also can be
used for fluorescent detection of a genetic sequence. Such
techniques have been described, for example, in U.S. Pat. Nos.
5,968,740 and 5,858,659. A probe also can be affixed to an
electrode surface for the electrochemical detection of nucleic acid
sequences such as described by Kayyem et al. U.S. Pat. No.
5,952,172 and by Kelley, S. O. et al. (1999) Nucleic Acids Res.
27:4830-4837.
[0049] The terms "protein", "polypeptide" and "peptide" are used
interchangeably herein when referring to a gene product.
[0050] The term "recombinant protein" refers to a polypeptide which
is produced by recombinant DNA techniques, wherein generally, DNA
encoding the polypeptide is inserted into a suitable expression
vector which is in turn used to transform a host cell to produce
the heterologous protein.
[0051] The term "treating" as used herein is intended to encompass
curing as well as ameliorating at least one symptom of the
condition or disease. For example, in the case of cancer, treatment
includes a reduction in cachexia. Evidence of treatment may be
clinical or subclinical.
[0052] As used herein, the term "vector" refers to a nucleic acid
molecule capable of transporting another nucleic acid to which it
has been linked. One type of preferred vector is an episome, i.e.,
a nucleic acid capable of extra-chromosomal replication. Preferred
vectors are those capable of autonomous replication and/or
expression of nucleic acids to which they are linked. Vectors
capable of directing the expression of genes to which they are
operatively linked are referred to herein as "expression vectors".
In general, expression vectors of utility in recombinant DNA
techniques are often in the form of "plasmids" which refer
generally to circular double stranded DNA loops which, in their
vector form are not bound to the chromosome. In the present
specification, "plasmid" and "vector" are used interchangeably as
the plasmid is the most commonly used form of vector. However, the
invention is intended to include such other forms of expression
vectors which serve equivalent functions and which become known in
the art subsequently hereto.
[0053] The term "wild-type allele" refers to an allele of a gene
which, when present in two copies in a subject results in a
wild-type phenotype. There can be several different wild-type
alleles of a specific gene, since certain nucleotide changes in a
gene may not affect the phenotype of a subject having two copies of
the gene with the nucleotide changes.
Nucleic Acids
[0054] In one aspect, the nucleic acid sequences of the gene's
allelic variants, or portions thereof, can be the basis for probes
or primers, e.g., in methods for determining the identity of the
allelic variant of the polymorphic region. Thus, they can be used
in the methods of the invention to determine whether a subject is
at risk of developing colorectal cancer.
[0055] The methods of the invention can use nucleic acids isolated
from vertebrates. In one aspect, the vertebrate nucleic acids are
mammalian nucleic acids. In a further aspect, the nucleic acids
used in the methods of the invention are a human nucleic acids.
[0056] Primers for use in the methods of the invention are nucleic
acids which hybridize to a nucleic acid sequence which is adjacent
to the region of interest (e.g., the 5'-untranslated region of the
TS gene) or which covers the region of interest and is extended. A
primer can be used alone in a detection method, or a primer can be
used together with at least one other primer or probe in a
detection method. Primers can also be used to amplify at least a
portion of a nucleic acid. Probes for use in the methods of the
invention are nucleic acids which hybridize to the region of
interest and which are not further extended. For example, a probe
is a nucleic acid which hybridizes to the polymorphic region of the
gene of interest, and which by hybridization or absence of
hybridization to the DNA of a subject will be indicative of the
identity of the allelic variant of the polymorphic region of the
gene of interest.
[0057] In one embodiment, primers comprise a nucleotide sequence
which comprises a region having a nucleotide sequence which
hybridizes under stringent conditions to about: 6, or alternatively
8, or alternatively 10, or alternatively 12, or alternatively 25,
or alternatively 30, or alternatively 40, or alternatively 50, or
alternatively 75 consecutive nucleotides of the gene of
interest.
[0058] Primers can be complementary to nucleotide sequences located
close to each other or further apart, depending on the use of the
amplified DNA. For example, primers can be chosen such that they
amplify DNA fragments of at least about 10 nucleotides or as much
as several kilobases. Preferably, the primers of the invention will
hybridize selectively to nucleotide sequences located about 150 to
about 350 nucleotides apart.
[0059] For amplifying at least a portion of a nucleic acid, a
forward primer (i.e., 5' primer) and a reverse primer (i.e., 3'
primer) will preferably be used. Forward and reverse primers
hybridize to complementary strands of a double stranded nucleic
acid, such that upon extension from each primer, a double stranded
nucleic acid is amplified.
[0060] Yet other preferred primers of the invention are nucleic
acids which are capable of selectively hybridizing to an allelic
variant of a polymorphic region of the gene of interest. Thus, such
primers can be specific for the gene of interest sequence, so long
as they have a nucleotide sequence which is capable of hybridizing
to the gene of interest.
[0061] The probe or primer may further comprises a label attached
thereto, which, e.g., is capable of being detected, e.g. the label
group is selected from amongst radioisotopes, fluorescent
compounds, enzymes, and enzyme co-factors.
[0062] Additionally, the isolated nucleic acids used as probes or
primers may be modified to become more stable. Exemplary nucleic
acid molecules which are modified include phosphoramidate,
phosphothioate and methylphosphonate analogs of DNA (see also U.S.
Pat. Nos. 5,176,996; 5,264,564; and 5,256,775).
[0063] The nucleic acids used in the methods of the invention can
also be modified at the base moiety, sugar moiety, or phosphate
backbone, for example, to improve stability of the molecule. The
nucleic acids, e.g., probes or primers, may include other appended
groups such as peptides (e.g., for targeting host cell receptors in
vivo), or agents facilitating transport across the cell membrane
(see, e.g., Letsinger et al., (1989) Proc. Natl. Acad. Sci. U.S.A.
86:6553-6556; Lemaitre et al., (1987) Proc. Natl. Acad. Sci.
84:648-652; and PCT Publication No. WO 88/09810, published Dec. 15,
1988), hybridization-triggered cleavage agents, (see, e.g., Krol et
al., (1988) BioTechniques 6:958-976) or intercalating agents (see,
e.g., Zon (1988) Pharm. Res. 5:539-549). To this end, the nucleic
acid used in the methods of the invention may be conjugated to
another molecule, e.g., a peptide, hybridization triggered
cross-linking agent, transport agent, hybridization-triggered
cleavage agent, etc.
[0064] The isolated nucleic acids used in the methods of the
invention can also comprise at least one modified sugar moiety
selected from the group including but not limited to arabinose,
2-fluoroarabinose, xylulose, and hexose or, alternatively, comprise
at least one modified phosphate backbone selected from the group
consisting of a phosphorothioate, a phosphorodithioate, a
phosphoramidothioate, a phosphoramidate, a phosphordiamidate, a
methylphosphonate, an alkyl phosphotriester, and a formacetal or
analog thereof.
[0065] The nucleic acids, or fragments thereof, to be used in the
methods of the invention can be prepared according to methods well
known in the art and described, e.g., in Sambrook et al. (1989)
supra. For example, discrete fragments of the DNA can be prepared
and cloned using restriction enzymes. Alternatively, discrete
fragments can be prepared using the Polymerase Chain Reaction (PCR)
using primers having an appropriate sequence under the
manufacturer's conditions.
[0066] Oligonucleotides can be synthesized by standard methods
known in the art, e.g. by use of an automated DNA synthesizer (such
as are commercially available from Biosearch, Applied Biosystems,
etc.). As examples, phosphorothioate oligonucleotides can be
synthesized by the method of Stein et al. (1988) Nucl. Acids Res.
16:3209, methylphosphonate oligonucleotides can be prepared by use
of controlled pore glass polymer supports (Sarin et al., (1988)
Proc. Natl. Acad. Sci. U.S.A. 85:7448-7451), etc.
Predictive Medicine and Pharmacogenomics
[0067] The invention further features predictive medicines, which
are based, at least in part, on determination of the identity of
the polymorphic region of the gene of interest.
[0068] For example, information obtained using the diagnostic
assays described herein is useful for determining if a subject will
respond to cancer treatment of a given type. Based on the
prognostic information, a doctor can recommend a regimen (e.g. diet
or exercise) or therapeutic protocol, useful for treating cancer in
the individual.
[0069] In addition, knowledge of the identity of a particular
allele in an individual (the gene profile) allows customization of
therapy for a particular disease to the individual's genetic
profile, the goal of "pharmacogenomics". For example, an
individual's genetic profile can enable a doctor: 1) to more
effectively prescribe a drug that will address the molecular basis
of the disease or condition; and 2) to better determine the
appropriate dosage of a particular drug. Expression patterns of
individual patients can then be compared to the expression profile
of the disease to determine the appropriate drug and dose to
administer to the patient.
[0070] The ability to target populations expected to show the
highest clinical benefit, based on the normal or disease genetic
profile, can enable: 1) the repositioning of marketed drugs with
disappointing market results; 2) the rescue of drug candidates
whose clinical development has been discontinued as a result of
safety or efficacy limitations, which are patient
subgroup-specific; and 3) an accelerated and less costly
development for drug candidates and more optimal drug labeling.
[0071] Detection of point mutations can be accomplished by
molecular cloning of the specified allele and subsequent sequencing
of that allele using techniques known in the art. Alternatively,
the gene sequences can be amplified directly from a genomic DNA
preparation from the tumor tissue using PCR, and the sequence
composition is determined from the amplified product. As described
more fully below, numerous methods are available for analyzing a
subject's DNA for mutations at a given genetic locus such as the
gene of interest.
[0072] A detection method is allele specific hybridization using
probes overlapping the polymorphic site and having about 5, or
alternatively 10, or alternatively 20, or alternatively 25, or
alternatively 30 nucleotides around the polymorphic region. In
another embodiment of the invention, several probes capable of
hybridizing specifically to the allelic variant are attached to a
solid phase support, e.g., a "chip". Oligonucleotides can be bound
to a solid support by a variety of processes, including
lithography. For example a chip can hold up to 250,000
oligonucleotides (GeneChip, Affymetrix). Mutation detection
analysis using these chips comprising oligonucleotides, also termed
"DNA probe arrays" is described e.g., in Cronin et al. (1996) Human
Mutation 7:244.
[0073] In other detection methods, it is necessary to first amplify
at least a portion of the gene of interest prior to identifying the
allelic variant. Amplification can be performed, e.g., by PCR
and/or LCR, according to methods known in the art. In one
embodiment, genomic DNA of a cell is exposed to two PCR primers and
amplification for a number of cycles sufficient to produce the
required amount of amplified DNA.
[0074] Alternative amplification methods include: self sustained
sequence replication (Guatelli, J. C. et al., (1990) Proc. Natl.
Acad. Sci. USA 87:1874-1878), transcriptional amplification system
(Kwoh, D. Y. et al., (1989) Proc. Natl. Acad. Sci. USA
86:1173-1177), Q-Beta Replicase (Lizardi, P. M. et al., (1988)
Bio/Technology 6:1197), or any other nucleic acid amplification
method, followed by the detection of the amplified molecules using
techniques known to those of skill in the art. These detection
schemes are useful for the detection of nucleic acid molecules if
such molecules are present in very low numbers.
[0075] In one embodiment, any of a variety of sequencing reactions
known in the art can be used to directly sequence at least a
portion of the gene of interest and detect allelic variants, e.g.,
mutations, by comparing the sequence of the sample sequence with
the corresponding wild-type (control) sequence. Exemplary
sequencing reactions include those based on techniques developed by
Maxam and Gilbert ((1997) Proc. Natl Acad Sci USA 74:560) or Sanger
(Sanger et al. (1977) Proc. Nat. Acad. Sci 74:5463). It is also
contemplated that any of a variety of automated sequencing
procedures can be utilized when performing the subject assays
(Biotechniques (1995) 19:448), including sequencing by mass
spectrometry (see, for example, U.S. Pat. No. 5,547,835 and
international patent application Publication Number WO94/16101,
entitled DNA Sequencing by Mass Spectrometry by H. Koster; U.S.
Pat. No. 5,547,835 and international patent application Publication
Number WO 94/21822 entitled "DNA Sequencing by Mass Spectrometry
Via Exonuclease Degradation" by H. Koster; U.S. Pat. No. 5,605,798
and International Patent Application No. PCT/US96/03651 entitled
DNA Diagnostics Based on Mass Spectrometry by H. Koster; Cohen et
al. (1996) Adv. Chromat. 36:127-162; and Griffin et al. (1993) Appl
Biochem Bio. 38:147-159). It will be evident to one skilled in the
art that, for certain embodiments, the occurrence of only one, two
or three of the nucleic acid bases need be determined in the
sequencing reaction. For instance, A-track or the like, e.g., where
only one nucleotide is detected, can be carried out.
[0076] Yet other sequencing methods are disclosed, e.g., in U.S.
Pat. No. 5,580,732 entitled "Method Of DNA Sequencing Employing A
Mixed DNA-Polymer Chain Probe" and U.S. Pat. No. 5,571,676 entitled
"Method For Mismatch-Directed In Vitro DNA Sequencing".
[0077] In some cases, the presence of the specific allele in DNA
from a subject can be shown by restriction enzyme analysis. For
example, the specific nucleotide polymorphism can result in a
nucleotide sequence comprising a restriction site which is absent
from the nucleotide sequence of another allelic variant.
[0078] In a further embodiment, protection from cleavage agents
(such as a nuclease, hydroxylamine or osmium tetroxide and with
piperidine) can be used to detect mismatched bases in RNA/RNA
DNA/DNA, or RNA/DNA heteroduplexes (see, e.g., Myers et al. (1985)
Science 230:1242). In general, the technique of "mismatch cleavage"
starts by providing heteroduplexes formed by hybridizing a control
nucleic acid, which is optionally labeled, e.g., RNA or DNA,
comprising a nucleotide sequence of the allelic variant of the gene
of interest with a sample nucleic acid, e.g., RNA or DNA, obtained
from a tissue sample. The double-stranded duplexes are treated with
an agent which cleaves single-stranded regions of the duplex such
as duplexes formed based on basepair mismatches between the control
and sample strands. For instance, RNA/DNA duplexes can be treated
with RNase and DNA/DNA hybrids treated with S1 nuclease to
enzymatically digest the mismatched regions. In other embodiments,
either DNA/DNA or RNA/DNA duplexes can be treated with
hydroxylamine or osmium tetroxide and with piperidine in order to
digest mismatched regions. After digestion of the mismatched
regions, the resulting material is then separated by size on
denaturing polyacrylamide gels to determine whether the control and
sample nucleic acids have an identical nucleotide sequence or in
which nucleotides they are different. See, for example, Cotton et
al. (1988) Proc. Natl. Acad. Sci. USA 85:4397; Saleeba et al.
(1992) Methods Enzy. 217:286-295. In another embodiment, the
control or sample nucleic acid is labeled for detection.
[0079] In other embodiments, alterations in electrophoretic
mobility is used to identify the particular allelic variant. For
example, single strand conformation polymorphism (SSCP) may be used
to detect differences in electrophoretic mobility between mutant
and wild type nucleic acids (Orita et al. (1989) Proc Natl. Acad.
Sci USA 86:2766, see also Cotton (1993) Mutat. Res. 285:125-144 and
Hayashi (1992) Genet Anal Tech Appl 9:73-79). Single-stranded DNA
fragments of sample and control nucleic acids are denatured and
allowed to renature. The secondary structure of single-stranded
nucleic acids varies according to sequence, the resulting
alteration in electrophoretic mobility enables the detection of
even a single base change. The DNA fragments may be labeled or
detected with labeled probes. The sensitivity of the assay may be
enhanced by using RNA (rather than DNA), in which the secondary
structure is more sensitive to a change in sequence. In another
preferred embodiment, the subject method utilizes heteroduplex
analysis to separate double stranded heteroduplex molecules on the
basis of changes in electrophoretic mobility (Keen et al. (1991)
Trends Genet. 7:5).
[0080] In yet another embodiment, the identity of the allelic
variant is obtained by analyzing the movement of a nucleic acid
comprising the polymorphic region in polyacrylamide gels containing
a gradient of denaturant, which is assayed using denaturing
gradient gel electrophoresis (DGGE) (Myers et al. (1985) Nature
313:495). When DGGE is used as the method of analysis, DNA will be
modified to insure that it does not completely denature, for
example by adding a GC clamp of approximately 40 bp of high-melting
GC-rich DNA by PCR. In a further embodiment, a temperature gradient
is used in place of a denaturing agent gradient to identify
differences in the mobility of control and sample DNA (Rosenbaum
and Reissner (1987) Biophys Chem 265:1275).
[0081] Examples of techniques for detecting differences of at least
one nucleotide between 2 nucleic acids include, but are not limited
to, selective oligonucleotide hybridization, selective
amplification, or selective primer extension. For example,
oligonucleotide probes may be prepared in which the known
polymorphic nucleotide is placed centrally (allele-specific probes)
and then hybridized to target DNA under conditions which permit
hybridization only if a perfect match is found (Saiki et al. (1986)
Nature 324:163); Saiki et al. (1989) Proc. Natl. Acad. Sci USA
86:6230; and Wallace et al. (1979) Nucl. Acids Res. 6:3543). Such
allele specific oligonucleotide hybridization techniques may be
used for the detection of the nucleotide changes in the
polylmorphic region of the gene of interest. For example,
oligonucleotides having the nucleotide sequence of the specific
allelic variant are attached to a hybridizing membrane and this
membrane is then hybridized with labeled sample nucleic acid.
Analysis of the hybridization signal will then reveal the identity
of the nucleotides of the sample nucleic acid.
[0082] Alternatively, allele specific amplification technology
which depends on selective PCR amplification may be used in
conjunction with the instant invention. Oligonucleotides used as
primers for specific amplification may carry the allelic variant of
interest in the center of the molecule (so that amplification
depends on differential hybridization) (Gibbs et al. (1989) Nucleic
Acids Res. 17:2437-2448) or at the extreme 3' end of one primer
where, under appropriate conditions, mismatch can prevent, or
reduce polymerase extension (Prossner (1993) Tibtech 11:238; Newton
et al. (1989) Nucl. Acids Res. 17:2503). This technique is also
termed "PROBE" for Probe Oligo Base Extension. In addition it may
be desirable to introduce a novel restriction site in the region of
the mutation to create cleavage-based detection (Gasparini et al.
(1992) Mol. Cell Probes 6:1).
[0083] In another embodiment, identification of the allelic variant
is carried out using an oligonucleotide ligation assay (OLA), as
described, e.g., in U.S. Pat. No. 4,998,617 and in Landegren, U. et
al. Science 241:1077-1080 (1988). The OLA protocol uses two
oligonucleotides which are designed to be capable of hybridizing to
abutting sequences of a single strand of a target. One of the
oligonucleotides is linked to a separation marker, e.g.,
biotinylated, and the other is detectably labeled. If the precise
complementary sequence is found in a target molecule, the
oligonucleotides will hybridize such that their termini abut, and
create a ligation substrate. Ligation then permits the labeled
oligonucleotide to be recovered using avidin, or another biotin
ligand. Nickerson, D. A. et al. have described a nucleic acid
detection assay that combines attributes of PCR and OLA (Nickerson,
D. A. et al. (1990) Proc. Natl. Acad. Sci. (U.S.A.) 87:8923-8927).
In this method, PCR is used to achieve the exponential
amplification of target DNA, which is then detected using OLA.
[0084] Several techniques based on this OLA method have been
developed and can be used to detect the specific allelic variant of
the polymorphic region of the gene of interest. For example, U.S.
Pat. No. 5,593,826 discloses an OLA using an oligonucleotide having
3'-amino group and a 5'-phosphorylated oligonucleotide to form a
conjugate having a phosphoramidate linkage. In another variation of
OLA described in Tobe et al. (1996) Nucleic Acids Res. 24: 3728),
OLA combined with PCR permits typing of two alleles in a single
microtiter well. By marking each of the allele-specific primers
with a unique hapten, i.e. digoxigenin and fluorescein, each OLA
reaction can be detected by using hapten specific antibodies that
are labeled with different enzyme reporters, alkaline phosphatase
or horseradish peroxidase. This system permits the detection of the
two alleles using a high throughput format that leads to the
production of two different colors.
[0085] The invention further provides methods for detecting the
single nucleotide polymorphism in the gene of interest. Because
single nucleotide polymorphisms constitute sites of variation
flanked by regions of invariant sequence, their analysis requires
no more than the determination of the identity of the single
nucleotide present at the site of variation and it is unnecessary
to determine a complete gene sequence for each patient. Several
methods have been developed to facilitate the analysis of such
single nucleotide polymorphisms.
[0086] In one embodiment, the single base polymorphism can be
detected by using a specialized exonuclease-resistant nucleotide,
as disclosed, e.g., in Mundy, C. R. (U.S. Pat. No. 4,656,127).
According to the method, a primer complementary to the allelic
sequence immediately 3' to the polymorphic site is permitted to
hybridize to a target molecule obtained from a particular animal or
human. If the polymorphic site on the target molecule contains a
nucleotide that is complementary to the particular
exonuclease-resistant nucleotide derivative present, then that
derivative will be incorporated onto the end of the hybridized
primer. Such incorporation renders the primer resistant to
exonuclease, and thereby permits its detection. Since the identity
of the exonuclease-resistant derivative of the sample is known, a
finding that the primer has become resistant to exonucleases
reveals that the nucleotide present in the polymorphic site of the
target molecule was complementary to that of the nucleotide
derivative used in the reaction. This method has the advantage that
it does not require the determination of large amounts of
extraneous sequence data.
[0087] In another embodiment of the invention, a solution-based
method is used for determining the identity of the nucleotide of
the polymorphic site. Cohen, D. et al. (French Patent 2,650,840;
PCT Appln. No. WO91/02087). As in the Mundy method of U.S. Pat. No.
4,656,127, a primer is employed that is complementary to allelic
sequences immediately 3' to a polymorphic site. The method
determines the identity of the nucleotide of that site using
labeled dideoxynucleotide derivatives, which, if complementary to
the nucleotide of the polymorphic site will become incorporated
onto the terminus of the primer.
[0088] An alternative method, known as Genetic Bit Analysis or
GBA.TM. is described by Goelet, P. et al. (PCT Appln. No.
92/15712). This method uses mixtures of labeled terminators and a
primer that is complementary to the sequence 3' to a polymorphic
site. The labeled terminator that is incorporated is thus
determined by, and complementary to, the nucleotide present in the
polymorphic site of the target molecule being evaluated. In
contrast to the method of Cohen et al. (French Patent 2,650,840;
PCT Appln. No. WO91/02087) the method of Goelet, P. et al. supra,
is preferably a heterogeneous phase assay, in which the primer or
the target molecule is immobilized to a solid phase.
[0089] Recently, several primer-guided nucleotide incorporation
procedures for assaying polymorphic sites in DNA have been
described (Komher, J. S. et al. (1989) Nucl. Acids. Res.
17:7779-7784; Sokolov, B. P. (1990) Nucl. Acids Res. 18:3671;
Syvanen, A.-C., et al. (1990) Genomics 8:684-692; Kuppuswamy, M. N.
et al. (1991) Proc. Natl. Acad. Sci. (U.S.A.) 88:1143-1147;
Prezant, T. R. et al. (1992) Hum. Mutat. 1:159-164; Ugozzoli, L. et
al. (1992) GATA 9:107-112; Nyren, P. et al. (1993) Anal. Biochem.
208:171-175). These methods differ from GBA.TM. in that they all
rely on the incorporation of labeled deoxynucleotides to
discriminate between bases at a polymorphic site. In such a format,
since the signal is proportional to the number of deoxynucleotides
incorporated, polymorphisms that occur in runs of the same
nucleotide can result in signals that are proportional to the
length of the run (Syvanen, A.-C., et al. (1993) Amer. J. Hum.
Genet. 52:46-59).
[0090] If the polymorphic region is located in the coding region of
the gene of interest, yet other methods than those described above
can be used for determining the identity of the allelic variant.
For example, identification of the allelic variant, which encodes a
mutated signal peptide, can be performed by using an antibody
specifically recognizing the mutant protein in, e.g.,
immunohistochemistry or immunoprecipitation. Antibodies to the
wild-type or signal peptide mutated forms of the signal peptide
proteins can be prepared according to methods known in the art.
[0091] Antibodies directed against wild type or mutant peptides
encoded by the allelic variants of the gene of interest may also be
used in disease diagnostics and prognostics. Such diagnostic
methods, may be used to detect abnormalities in the level of
expression of the peptide, or abnormalities in the structure and/or
tissue, cellular, or subcellular location of the peptide. Protein
from the tissue or cell type to be analyzed may easily be detected
or isolated using techniques which are well known to one of skill
in the art, including but not limited to Western blot analysis. For
a detailed explanation of methods for carrying out Western blot
analysis, see Sambrook et al., (1989) supra, at Chapter 18. The
protein detection and isolation methods employed herein can also be
such as those described in Harlow and Lane, (1988) supra. This can
be accomplished, for example, by immunofluorescence techniques
employing a fluorescently labeled antibody (see below) coupled with
light microscopic, flow cytometric, or fluorimetric detection. The
antibodies (or fragments thereof) useful in the present invention
may, additionally, be employed histologically, as in
immunofluorescence or immunoelectron microscopy, for in situ
detection of the peptides or their allelic variants. In situ
detection may be accomplished by removing a histological specimen
from a patient, and applying thereto a labeled antibody of the
present invention. The antibody (or fragment) is preferably applied
by overlaying the labeled antibody (or fragment) onto a biological
sample. Through the use of such a procedure, it is possible to
determine not only the presence of the subject polypeptide, but
also its distribution in the examined tissue. Using the present
invention, one of ordinary skill will readily perceive that any of
a wide variety of histological methods (such as staining
procedures) can be modified in order to achieve such in situ
detection.
[0092] Often a solid phase support or carrier is used as a support
capable of binding an antigen or an antibody. Well-known supports
or carriers include glass, polystyrene, polypropylene,
polyethylene, dextran, nylon, amylases, natural and modified
celluloses, polyacrylamides, gabbros, and magnetite. The nature of
the carrier can be either soluble to some extent or insoluble for
the purposes of the present invention. The support material may
have virtually any possible structural configuration so long as the
coupled molecule is capable of binding to an antigen or antibody.
Thus, the support configuration may be spherical, as in a bead, or
cylindrical, as in the inside surface of a test tube, or the
external surface of a rod. Alternatively, the surface may be flat
such as a sheet, test strip, etc. or alternatively polystyrene
beads. Those skilled in the art will know many other suitable
carriers for binding antibody or antigen, or will be able to
ascertain the same by use of routine experimentation.
[0093] Moreover, it will be understood that any of the above
methods for detecting alterations in a gene or gene product or
polymorphic variants can be used to monitor the course of treatment
or therapy.
[0094] The methods described herein may be performed, for example,
by utilizing pre-packaged diagnostic kits, such as those described
below, comprising at least one probe or primer nucleic acid
described herein, which may be conveniently used, e.g., to
determine whether a subject has or is at risk of developing
colorectal cancer.
[0095] Sample nucleic acid for use in the above-described
diagnostic and prognostic methods can be obtained from any cell
type or tissue of a subject. For example, a subject's bodily fluid
(e.g. blood) can be obtained by known techniques (e.g.,
venipuncture). Alternatively, nucleic acid tests can be performed
on dry samples (e.g., hair or skin). Fetal nucleic acid samples can
be obtained from maternal blood as described in International
Patent Application No. WO91/07660 to Bianchi. Alternatively,
amniocytes or chorionic villi can be obtained for performing
prenatal testing.
[0096] Diagnostic procedures can also be performed in situ directly
upon tissue sections (fixed and/or frozen) of patient tissue
obtained from biopsies or resections, such that no nucleic acid
purification is necessary. Nucleic acid reagents can be used as
probes and/or primers for such in situ procedures (see, for
example, Nuovo, G. J. (1992) "PCR In Situ Hybridization: Protocols
And Applications", Raven Press, NY).
[0097] In addition to methods which focus primarily on the
detection of one nucleic acid sequence, profiles can also be
assessed in such detection schemes. Fingerprint profiles can be
generated, for example, by utilizing a differential display
procedure, Northern analysis and/or RT-PCR.
Methods of Treatment
[0098] The invention further provides methods of treating subjects
having cancer. In one embodiment, the method comprises (a)
determining the identity of the allelic variant; and (b)
administering to the subject an effective amount of a compound that
provides therapeutic benefits for the specific allelic variant.
Kits
[0099] As set forth herein, the invention provides methods, e.g.,
diagnostic and therapeutic methods, e.g., for determining the type
of allelic variant of a polymorphic region present in the gene of
interest, such as a human TS gene. In some embodiments, the methods
use probes or primers comprising nucleotide sequences which are
complementary to the polymorphic region of the gene of interest.
Accordingly, the invention provides kits for performing these
methods.
[0100] In an embodiment, the invention provides a kit for
determining whether a subject responds to cancer treatment or
alternatively one of various treatment options. The kits contain
one of more of the compositions described above and instructions
for use. As an example only, the invention also provides kits for
determining response to cancer treatment containing a first and a
second oligonucleotide specific for the polymorphic region of the
TS gene, namely in the 5'-untranslated region. Oligonucleotides
"specific for" a genetic locus bind either to the polymorphic
region of the locus or bind adjacent to the polymorphic region of
the locus. For oligonucleotides that are to be used as primers for
amplification, primers are adjacent if they are sufficiently close
to be used to produce a polynucleotide comprising the polymorphic
region. In one embodiment, oligonucleotides are adjacent if they
bind within about 1-2 kb, and preferably less than 1 kb from the
polymorphism. Specific oligonucleotides are capable of hybridizing
to a sequence, and under suitable conditions will not bind to a
sequence differing by a single nucleotide.
[0101] The kit can comprise at least one probe or primer which is
capable of specifically hybridizing to the polymorphic region of
the gene of interest and instructions for use. The kits preferably
comprise at least one of the above described nucleic acids.
Preferred kits for amplifying at least a portion of the gene of
interest comprise two primers, at least one of which is capable of
hybridizing to the allelic variant sequence. Such kits are suitable
for detection of genotype by, for example, fluorescence detection,
by electrochemical detection, or by other detection.
[0102] Oligonucleotides, whether used as probes or primers,
contained in a kit can be detectably labeled. Labels can be
detected either directly, for example for fluorescent labels, or
indirectly. Indirect detection can include any detection method
known to one of skill in the art, including biotin-avidin
interactions, antibody binding and the like. Fluorescently labeled
oligonucleotides also can contain a quenching molecule.
Oligonucleotides can be bound to a surface. In one embodiment, the
preferred surface is silica or glass. In another embodiment, the
surface is a metal electrode.
[0103] Yet other kits of the invention comprise at least one
reagent necessary to perform the assay. For example, the kit can
comprise an enzyme. Alternatively the kit can comprise a buffer or
any other necessary reagent.
[0104] Conditions for incubating a nucleic acid probe with a test
sample depend on the format employed in the assay, the detection
methods used, and the type and nature of the nucleic acid probe
used in the assay. One skilled in the art will recognize that any
one of the commonly available hybridization, amplification or
immunological assay formats can readily be adapted to employ the
nucleic acid probes for use in the present invention. Examples of
such assays can be found in Chard, T. (1986) "An Introduction to
Radioimmunoassay and Related Techniques" Elsevier Science
Publishers, Amsterdam, The Netherlands; Bullock, G. R. et al.,
"Techniques in Immunocytochemistry" Academic Press, Orlando, Fla.
Vol. 1 (1982), Vol. 2 (1983), Vol. 3 (1985); Tijssen, P., (1985)
"Practice and Theory of Immunoassays: Laboratory Techniques in
Biochemistry and Molecular Biology", Elsevier Science Publishers,
Amsterdam, The Netherlands.
[0105] The test samples used in the diagnostic kits include cells,
protein or membrane extracts of cells, or biological fluids such as
sputum, blood, serum, plasma, or urine. The test sample used in the
above-described method will vary based on the assay format, nature
of the detection method and the tissues, cells or extracts used as
the sample to be assayed. Methods for preparing protein extracts or
membrane extracts of cells are known in the art and can be readily
adapted in order to obtain a sample which is compatible with the
system utilized.
[0106] The kits can include all or some of the positive controls,
negative controls, reagents, primers, sequencing markers, probes
and antibodies described herein for determining the subject's
genotype in the polymorphic region of the gene of interest.
[0107] As amenable, these suggested kit components may be packaged
in a manner customary for use by those of skill in the art. For
example, these suggested kit components may be provided in solution
or as a liquid dispersion or the like.
Other Uses for the Nucleic Acids of the Invention
[0108] The identification of the allele of the gene of interest can
also be useful for identifying an individual among other
individuals from the same species. For example, DNA sequences can
be used as a fingerprint for detection of different individuals
within the same species (Thompson, J. S. and Thompson, eds., (1991)
"Genetics in Medicine", W B Saunders Co., Philadelphia, Pa.). This
is useful, e.g., in forensic studies.
[0109] The present invention is further illustrated by the
following examples which should not be construed as limiting in any
way. The contents of all cited references (including literature
references, issued patents, published patent applications as cited
throughout this application are hereby expressly incorporated by
reference. The practice of the present invention will employ,
unless otherwise indicated, conventional techniques of cell
biology, cell culture, molecular biology, transgenic biology,
microbiology, recombinant DNA, and immunology, which are within the
skill of the art. Such techniques are explained fully in the
literature. See, for example, Sambrook, et al., (1989)
[0110] The invention now being generally described, it will be more
readily understood by reference to the following examples which are
included merely for purposes of illustration of certain aspects and
embodiments of the present invention, and are not intended to limit
the invention.
EXAMPLES
Example 1
Gene Expression Profile in Normal Tissue Predicts Pelvic Recurrence
in Patients with Rectal Cancer Treated with Adjuvant Chemoradiation
Therapy
[0111] The incidence of colorectal cancer has been rising within
the last decade and is now as high as 41,000 estimated new cases
and 8,500 deaths in the USA per year. (Jemal A. et al. (2002) CA
Cancer J Clin 52:23-47). In stage rectal cancer, local recurrence
occurs in 20-70% of patients treated with surgery alone. (Kapiteijn
E. et al. (2001) N. Engl. J. Med. 345:638-646). Adjuvant
radio-chemotherapy has been generally accepted in the United States
as standard therapy for patients who had surgical resection for
high-risk rectal cancer, (NTH Consensus Conference. Adjuvant
Therapy For Patients With Colon And Rectal Cancer (1990) JAMA
264:1444-1450).
[0112] Previous studies in rectal cancer showed associations
between elevated levels of certain genes, including TS and DPD (?).
(Salonga D. et al. (2000) Clin. Cancer Res. 6:1322-1327 and
Ishikawa et al. (1999) Clin. Cancer Res. 5:883-889) and a worse
outcome after neoadjuvant treatment with 5-FU.
[0113] Thus, it would be useful to identify a maker that would
predict pelvic recurrence in patients with rectal cancer treated
with adjuvant chemo-radiation. To this end, mRNA levels of putative
response determinant genes in tumor plus adjacent normal tissues
were measured. The investigated genes were involved in the 5-FU
pathway (TS, DPD), in DNA repair (ERCC1, RAD51), and angiogenesis
(VEGF).
[0114] Methods: Seventy-three (73) patients with locally advanced
rectal cancer (UICC stage II and III) were selected. They had been
previously treated with adjuvant pelvic irradiation (45 Gy) to the
whole pelvis with an additional boost up to 54 Gy plus 5-FU
infusion therapy following tumor resection tissue acquisition
during surgery. QRT-PCR (a fluorescence-based, quantitative
real-time detection method (Taqman.RTM.)) was performed on RNA
extracted from formalin-fixed, paraffin-embedded,
laser-capture-microdissected tissue to establish gene expression
levels. The mRNA was reverse transcribed to cDNA and the genes of
interest were quantified as well as an internal reference gene
(.beta.-Actin) as a control. All gene expression levels were
log-transformed prior to analysis. The maximal c.sup.2 method of
Miller, Siegmund (Miller R. and Siegmund D. Biometrics (1982)
38:1011-1016) and Halpern (Halpern J. (1982) Biometrics
38:1017-1023) was used to determine which gene expression best
segregated patients into poor- and good-prognosis subgoups 2000
bootstrap-like simulations were used to estimate the distribution
of the maximal c.sup.2 statistics under the null hypothesis of no
association.
TABLE-US-00001 TABLE 1 Time to local recurrence in rectal cancer
based on demographic and clinical parameters Probability .+-. SE of
Median time to recurrence Relative Risk Parameter n 5-yr recurrence
Month 95% CI Risk 95% CI p.sup.a Total Patients 73 0.53 .+-. 0.08
57.0 38.4, 130.2+.sup.b Age <50 years 30 0.48 .+-. 0.11 65.7
27.3, 130.2+ 1.00 Reference .gtoreq.50 years 43 0.57 .+-. 0.11 56.0
25.8, 124.6+ 1.24 0.60-2.56 0.56 Sex Male 48 0.58 .+-. 0.10 56.0
27.3, 124.6+ 1.00 Reference Female 25 0.44 .+-. 0.13 130.2+ 36.0,
130.2+ 0.62 0.28-1.40 0.24 Race Caucasian 50 0.57 .+-. 0.09 56.0
25.4, 124.6+ 1.00 Reference Other 23 0.43 .+-. 0.14 65.7 40.5,
130.2+ 0.63 0.28-1.40 0.25 pT pT.sub.2 22 0.61 .+-. 0.14 56.0 25.8,
130.2+ 1.00 Reference pT.sub.3 51 0.48 .+-. 0.09 65.7 38.4, 102.7+
0.96 0.45-2.04 0.91 pN pN.sub.0 35 0.43 .+-. 0.11 65.7 40.5, 130.2+
1.00 Reference pN.sub.+ 38 0.60 .+-. 0.10 56.0 25.8, 124.6+ 1.31
0.64-2.67 0.46 Grade I-II 57 0.60 .+-. 0.09 56.0 27.3, 130.2+ 1.00
Reference III 16 0.24 .+-. 0.12 103.7+ 65.7, 103.7+ 0.56 0.19-1.59
0.26 Surgery Type APR.sup.c 20 0.73 .+-. 0.15 56.0 25.2, 130.2+
1.00 Reference LAR 44 0.48 .+-. 0.09 65.7 38.4, 102.7+ 0.82
0.37-1.80 0.66 TRA 9 0.22 .+-. 0.14 103.7+ 103.7+, 103.7+ 0.51
0.11-2.35 .sup.aBased on Log-rank test. .sup.bThe estimates were
not reached. .sup.cAPR, abdominal perineal resection; LAR, lower
anterior resection; TRA, transanal resection
[0115] Results: Intra-tumoral mRNA levels of genes associated with
the 5-FU metabolism and DNA repair were not associated with the
outcome after adjuvant radio-chemotherapy in patients with rectal
cancer. Gene expression levels of TS, DPD, ERCC1, and VEGF in the
tumor adjacent normal tissue were associated with the clinical
outcome and can be useful to identify patients at higher risk for
pelvic recurrence. These results show that the gene expression of
the tumor adjacent normal tissue but not of the tumor tissue is
representative for the biological behavior of the tumor cells,
remaining after surgery, that may cause local tumor recurrence.
Example 2
TS Polymorphism in the Promoter Region Predicts Pelvic Recurrence
in Treated Cancer Patients
[0116] The enzyme thymidylate synthase (TS) catalyzes the
intracellular conversion of deoxyuridylate to deoxythymidylate
which is the sole de novo source of thymidylate, an essential
precursor for DNA synthesis. (Heidelberger C. et al. (1957) Nature
(179):663-666). It has been shown that the human thymidylate
synthase gene (hTS) is polymorphic with either double or triple
tandem repeats of a 28 base-pair sequence downstream of the
cap-site in the 5' terminal regulatory region. (Horie N. et al.
(1995) Cell Struct. Funct. 20:191-197).
[0117] A polymorphism in the TS gene and its correlation with the
efficacy of treatment with 5-FU was previously described in
co-owned U.S. patent application Ser. No. 09/715,764, (the entire
contents of which are incorporated by reference herein.) The
predictive polymorphism reported in this disclosure is a tandemly
repeated 28 base pair sequence in the thymidilate synthase gene's
5' UTR. Patients less likely to be responsive to treatment with a
TS directed drug, e.g., 5-fluorouracil, were determined to be
homozygous for this triple repeat of the tandemly repeated
sequence. Patients exhibiting heterozygous genotype for a double
repeat and a triple repeat of the tandemly repeated sequence. The
patients most likely to respond to administration of a TS directed
drug (e.g., 5-fluorouracil) are homozygous for a double repeat of
the tandemly repeated sequence.
[0118] Even after successful treatment, local recurrence for
patients with rectal cancer is a significant medical issue.
Patients with localized rectal cancer are treated with radiation
therapy to reduce the risk for local recurrence. The standard
therapy for locally advanced rectal cancer either pre or
postoperative is 5-FU chemotherapy and radiation therapy. Depending
on the pathological staging the risk for tumor recurrence is
between 10 and 60%. Identifying patients at high risk for tumor
recurrence will allow the development of better treatment
strategies for high risk patients. To this end, the 28 bp tandemly
repeat polymorphism in the TS gene was found to be predictive of
the risk of local recurrence in patients with rectal cancer treated
either with pre or postoperative chemoradiation therapy is
disclosed.
[0119] Methods: Forty-three (43) patients with locally advanced
rectal cancer, who were treated with either pre-operative or
post-operative 5-FU and pelvic radiation were analyzed. Genomic
DNAs were extracted from paraffin-embedded tissue samples.
Patients' genotype for the TS polymorphism was determined by
polymerase chain reaction (PCR) amplification of TS promoter
region. The PCR products were then electrophoresed, revealing bands
of 220 bp (2/2), 248 bp (3/3) or both (2/3). The genotyping was
repeated performed among 24 patients who developed local recurrence
and 19 patients who did not.
[0120] Results: Pelvic recurrences were found in 87% patients
homozygous for the triple tandemly repeated (3/3) genotype,
compared to 37% patients heterozygous with (2/2) and (2/3), after
either pre-operative or post-operative chemoradiation. P value is
less than 0.01. However, the (3/3) genotype was not associated with
advanced T or N stage, high grade histology, positive margin, or
vascular space invasion, thus is an independent predictor for
pelvic recurrence.
[0121] Thus, rectal cancer patients with 3/3 TS polymorphism are
less likely to be controlled locally after combined 5-FU and pelvic
radiation because of their resistance to both 5-FU and radiation.
Other chemotherapeutic agents such as CPT-11 or oxaliplatin, in
combination with radiation are alternatives therapies.
Example 3
TS 3' Polymorphism for Predicting Response and Survival to 5-FU and
Oxaliplatin
[0122] This example shows that a polymorphism associated with the
TS gene is associated with clinical response and survival to
5-FU/oxaliplatin chemotherapy in patients with cancer. Example 2,
supra, reports that the polymorphism in the TS promoter is
associated with TS gene expression in the normal tissue and the
tumor tissue. The findings indicate that it is possible to predict
TS gene expression in the tumor by measuring the TS polymorphism in
peripheral blood cells. Recently a polymorphism has been described
in the 3' end of the gene which have found to be associated with
intratumoral gene expression. This TS polymorphism is associated
with overall survival in patients treated with oxaliplatin and 5-FU
and is an independent predictor of outcome.
[0123] Prediction of response to 5-FU based chemotherapy and
prediction of optimal dose of 5-FU will maximize therapeutic
benefits and minimize treatment risks. Polymorphisms of genes
involved with the target of anticancer drugs and metabolism of
anti-cancer drugs can be predictive of intra-tumoral gene
expression levels. Polymorphism profiles can therefore influence
the selection or dosing of chemotherapeutic drugs. While not
wishing to be bound by any theory, the results reported herein also
explain the differences in toxicities and efficacy of anticancer
drugs in different ethnic groups since most of these polymorphisms
have been shown to have ethnic group associated characteristic gene
frequencies.
[0124] Methods: To investigate the functional relevance of this
polymorphism, the relative TS mRNA level and the polymorphism in
the 3'-untranslated region of the TS gene in 102 patients with
advanced colorectal carcinoma treated with 5-FU and oxaliplatin in
second or third line chemotherapy was evaluated. A polymerase chain
reaction amplification/RFLP analysis was performed to identify the
TS genotype using known methods known in the art. TS mRNA was
quantitated using a quantitative RT-PCR method known in the art and
described in Hankoshi T. et al. (1992) Cancer Res. 52:108-116.
[0125] The wildtype variant (+6 bp/+6 bp) was associated with
highest TS mRNA expression in the tumor (11.35, 95% CI: 6.43,
20.03) when compared to the heterozygous variant (+6 bp/-6 bp) with
a TS level of 5.42 (95% CI: 3.57, 8.24) and the homozygous mutant
variant with TS 2.71 (95% CI: 1.18, 5.26) (p=0.017, F-Test, see
Table 1).
TABLE-US-00002 TABLE 2 TS Genotype and TS mRNA Levels in Tumor
Tissue Comparison of TS TS TS Means Tissue Genotype N % Mean.sup.1
95% CI.sup.2 Genotype p- value.sup.3 Metastatic tumor tissue
+6bp/+6bp 13 30% 11.35 (6.43, 20.03) +6bp/+6bp vs. 0.007 (N = 43)
-6bp/-6bp +6bp/-6bp 24 56% 5.42 (3.57, 8.24) +6bp/+6bp vs. 0.041
+6bp/-6bp -6bp/-6bp 6 14% 2.71 (1.18, 6.26) +6bp/-6bp vs. 0.14
-6bp/-6bp Overall 0.017 .sup.1TS mean = geometric mean of mRNA
expression of TS relative to .beta. actin mRNA .sup.295% confidence
interval .sup.3p-value for the overall comparison is based on the
F-test, all other p-values are base on the LSD-Test (Least
significant difference test).
[0126] The wildtype variant (+6 bp/+6 bp) was associated with a
significant survival benefit when compared to the heterozygous
variant (+6 bp/-6 bp) and the homozygous mutant variant (p=0.040
based on the cox proportional hazards model stratified by ECOG and
multivariate analysis). Thus, this polymorphism in the 3'
untranslated region is predictive of clinical response and outcome
for some patients.
Example 4
ERCC1 Gene Polymorphism for Predicting Response and Survival to
5-FU/Oxaliplatin Chemotherapy
[0127] The results shown below establish that polymorphism
associated with the ERCC1 (excision repair cross complementation
group 1) gene is associated with clinical response and survival to
5-FU/oxaliplatin chemotherapy in patients with cancer and with
ERCC1 mRNA levels.
[0128] ERCC1 is a highly conserved enzyme, is specific to the
nucleotide excision repair (NER)1 pathway and its absence is
incompatible with life. Among the proteins involved in the NER, a
defect in the ERCC1 seems to be associated with the most severe DNA
repair deficiency.
[0129] Platinum compounds are becoming mainstay chemotherapy
treatment for gastric, ovarian, and colorectal cancer, among
others. Among its mechanisms of resistance, increased DNA repair
seems to be the most important mechanism.
[0130] Studies have shown that increased ERCC1 mRNA levels are
directly related to clinical resistance to cisplatin in human
ovarian cancer as well as cervical cancer. It has previously been
shown that ERCC1 mRNA levels are also directly correlated to
clinical resistance to 5-FU and cisplatin in gastric cancer
patients. It has also recently been shown that intra-tumoral ERCC1
mRNA levels are able to predict clinical response and overall
survival in patients with metatastatic colorectal cancer treated
with 5-FU/oxaliplatin.
[0131] The ERCC1 gene contains a very common polymorphism at codon
118 (exon 4). This polymorphism is a single nucleotide change
C.fwdarw.T which results in the same amino acid, asparagines. This
change converts a codon of common usage (AAC) to a less used codon
(AAT). The reported usage frequency of the latter is two-fold less
than the former. A study using ovarian cancer cell lines showed a
50% reduction in DNA adduct repair in a cell line containing the
polymorphism compared to the "wild-type." However, they were found
to be equally resistant to platinum.
[0132] In this study the ERCC1 polymorphism at codon 118 and
intra-tumoral ERCC1 mRNA levels of 32 patients with metastatic
colorectal cancer treated with 5-FU/oxaliplatin was assessed. The
median mRNA level was 2.95. Three of eleven (27.3%) patients with
the C/C genotype had ERCC1 mRNA levels greater than 2.95, whereas 5
out of 12 (41.7%) and 7 out of 9 (77.8%) of patients with the C/T
and T/T genotype respectively. When the mRNA levels of patients
containing the C allele was compared to those without the C allele,
the difference was statistically significant (p=0.049).
[0133] In a related study, the ERCC1 polymorphism at codon 118 and
the overall survival of 60 patients with metastatic colorectal
cancer treated with 5-FU/oxaliplatin was also assessed. The median
survival of patients was 531 days for those with the C/C genotype,
254 days for the CT genotype, and 256 days for the DT genotype
(trend p=0.089). The relative risk ratio for death was 2.12 for the
C/T and 2.36 for the T/T genotype. The median survival of patients
containing the T allele was 256 days and those without was 531 days
(p=0.056).
[0134] A search of the literature failed to provide an explanation
of how a "silent" polymorphism that results in a codon of lesser
usage can be associated with higher levels of mRNA. Without being
bound by any theory, Applicants note that this polymorphism is
associated with ERCC1 mRNA levels and therefore can predict
survival in patients with metastatic colorectal cancer treated with
5-FU/oxaliplatin.
Example 5
XPD (ERC2) Gene--Polymorphism for Predicting Response and Survival
to Platinum Based Chemotherapy
[0135] The results shown below establish that polymorphism
associated with the XPD gene is associated with clinical response
and survival to platinum based chemotherapy in patients with
cancer.
[0136] The XPD protein is essential in transcription and a major
participant in the nucleotide excision repair (NER) pathway.
Several polymorphisms in the XPD gene have been identified.
However, their functional sionilicance has not been elucidated. A
single nucleotide polymorphism in codon 751 (A.fwdarw.C) causes an
amino acid change Lys.fwdarw.Gln. There is evidence that the
polymorphism at codon 751 may affect DNA repair capability,
although previous studies regarding this issue have shown
conflicting results. (See Heidelberger, C. et al. (1957) Nature
179:663-666 and Hone, N. et al. (1995) Cell Struct. Funct.
20:191-197).
[0137] Increased DNA repair is a well-established mechanism for
chemo-resistance to platinum based compounds such as oxaliplatin.
The results reported herein show that increased gene expression of
ERCC1 (a member of the NER enzyme family) is associated with
resistance to 5-FU and cisplatin chemotherapy in gastric cancer
patients.
[0138] Methods: The XPD codon 751 polymorphic status of 69 patients
with metastatic colorectal cancer who previously had failed 5-FU
based chemotherapy and determined their response and overall
survival to 5-FU/oxaliplatin combination treatment. Genotyping was
done on white blood cells using the RFLP-PCR method.
[0139] Sixty seven patients were evaluated for response. The
overall response rate was 15% (10/67). 25% (5/20) patients with the
Lys/Lys genotype responded, compared to 11% (4/37) and 10% (1/10)
of those with the Lys/Gln and Gln/Gln genotypes respectively
(p=0.007 Fisher's exact test, two-tailed). More significantly,
among those with the Gln/Gln genotype, 50% (5/10) had progressive
disease compared to 10% (2/20) and 5% (2/37) of patients with the
Lys/Lys and Lys/Gln genotypes respectively.
[0140] The overall survival and its relation to the polymorphism
was also evaluated. For patients with the Lys/Lys genotype the
median survival was 530 days. Those with the Lys/Gln genotype had a
median survival of 356 days. Finally, those with the Gln/Gln
genotype had a median survival of 186 days (p=0.06 for trend).
Thus, the R.R. of death was 1.00 of the Lys/Lys group, 1.49 for
those with Lys/Gln, and 3.01 for the with Gln/Gln.
[0141] Results: The mechanism through which the Lys751Gln
polymorphism of the XPD gene affects DNA repair capacity and
resistance to chemotherapy is unknown. In fact, its very role in
DNA repair capacity is still being debated. Studies have shown
conflicting results on whether the polymorphism is associated with
increased or decreased DNA repair capacity. (See Heidelberger, C.
et al. (1957) Nature 179:663-666 and Horie, N. et al. (1995) Cell
Struct. Funct. 20:191-197).
[0142] These results show the XPD gene plays an important role in
chemo-resistance and genotyping. The 751 polymorphism is useful in
the prediction of clinical response, survival and clinical toxicity
to platinum based chemotherapy, as well as the design of novel
agents that modulate XPD function. XPD is also an important target
for drug development.
Example 6
XRCC1 Polymorphism is Predictive of Response in Patients Treated
with Platinum-Based Chemotherapy
[0143] Recently, Divine et al. (Proceedings AACR Annual meeting
March 2000, page 591) demonstrated that XRCC-1 polymorphism are
associated with higher AFB1-adducts and GPA somatic mutations but
also associated with lung cancer risk, colon cancer risk in
Egyptian (Abdel-Rahman et al. Proceedings AACR, Annual Meeting,
March 2000, page 595) and prostate cancer risk (Hu et al.
Proceedings AACR Annual Meeting, March 2000, page 596). A
polymorphism in exon 6 has been shown to have a protective effect
against bladder cancer development (Stem et al., Proceedings, AACR
Annual meeting March 2000, page 592).
[0144] XRCC-1 plays a central role in single strand break repair
and base excision repair. In addition, at least one of the gene
products required for single strand break repair in mammals, the
XRCC1 polypeptide is required for viability in mice, mutant cells
lacking XRCC-1 display cellular sensitivity to ionizing radiation
and alkylating agents and exhibit elevated spontaneous frequencies
of chromosome aberration. (Caldecott et al., Proceedings AACR
Annual Meeting, March 2000, page 891).
[0145] Methods: Forty-five (45) patients with advanced colorectal
cancer patients with 5-FU and oxaliplatin who failed at least one
prior chemotherapy regimen were selected. XRCC-1 polymorphisms and
their association with clinical outcome in patients with metastatic
colorectal cancer treated with 5-FU and oxaliplatin were studied.
These patients were heavily pretreated but received a platinum
compound for the first time. To determine whether variation in the
XRCC-1 DNA repair genes is related to host DNA damage, the
association between polymorphisms in XRCC1 (codon 399) and sister
chromatid exchange (SCE) frequencies (n=76) and polyphenol DNA
adducts (n=61) was studied. XRCC1 genotype was identified using
PCR-RFLP.
[0146] Results: From 45 patients, 6 patients (13%) underwent a
partial response, 30 patients (67%) had stable disease and 9 (20%)
had progressive disease. 18 patients had an A/A polymorphism, 22 an
A/G and 5 a G/G polymorphism. From the 6 responders, 5 have had an
A/A polymorphism and one an A/G polymorphism. 3 from 9 patients
with progressive disease had a G/G polymorphism and 4 from these 9
had an A/G polymorphism. Using the Jonckheere-Terpstra Test (monte
carlo two sided test) the p value was statistically significant
with 0.0063 with the 99% confidence interval of 0.0043 and 0.0083.
These data demonstrate that the A/A polymorphism is associated with
response to chemotherapy and patients with a G/G polymorphism are
associated with resistance to platinum compounds.
[0147] Mean SCE frequencies among current smokers who were
homozygous carriers of the 399Gln allele in XRCC1 were greater than
those in 399Arg/Arg current smokers. A possible gene-dosage effect
for XRCC1 399Gln and detectable DNA adducts was described, and
significantly more adducts among older subjects who were carriers
of the 399Gln allele than in younger subjects with the 399Arg/Arg
genotype suggesting that carriers of the polymorphic XRCC1 399Gln
allele may be at greater risk for DNA damage, (Carcinogenesis
(Oxford) 21(5)(2000):965-971).
[0148] Applicants have discovered that the XRCC-1 gene predicts
response in patients treated with platinum-based chemotherapy.
Identification of XRCC-1 polymorphism allowed not only to decide
whether platinum will have benefit but also may determine the risk
of side effects with platinum. XRCC-1 polymorphism could allow a
personalized approach to therapy--individualization of the dose and
choice of the anticancer drug based on use of this pre-screen. The
studies reported herein also identify individuals likely who have
benefit from platinum based chemotherapy and likely to experience
side effects of platinum agents.
Example 7
Human Glutathione S-Transferase P1 Polymorphism is Predictive of
Survival of Patients with Advanced Colorecal Cancer Treated with
5-FU/Oxaliplatin Chemotherapy
[0149] Glutathione transferases consist of a super-family of phase
III metabolic enzymes that catalyze the conjugation of reduced
glutathione. The detoxifying character of these reactions is
responsible for the protection of cellular macromolecules from
damage caused by carcinogenic and cytotoxic agents. (See Mannervik,
B. (1985) Adv. Enzymol. 57:357-417). GSTP1-1 has been shown to be
widely expressed in human epithelial tissues and to be
over-expressed in several tumors including colon tumors. (Terrier
P. et al. (1990) Am. J. Pathol. 137:845-853; Moscow J. A. et al.
(1989) Cancer Res. 49:1422-1428; Howie A. F. et al. (1990)
Carcinogenesis 11:451-458; Peters W. M. et al. (1992)
Gastroenterology 103:448-455; and Singh S. V. et al. (1990) Cancer
Lett. 51:43-48). Increased levels in tumors may be in part
responsible for the observed resistance to chemotherapy as it has
been found in several tumors, but the mechanism still remains
unknown. (Tsuchida S. et al. (1992) Rev. Biochem. Mol. Biol.
27:337-384). Factors that influence the expression level of GSTP1
may become important tools to predict therapy response and survival
of patients treated with certain drugs or drug combinations. A
G.fwdarw.A transition in exon 5 at nucleotide 313 leads to an amino
acid exchange in the protein from isoleucin to valine; as
previously reported by Board et al. (1989) Ann. Hum. Genet.
53:205-213. In-vitro cDNA expression studies revealed an
association between this amino acid change and a reduced activity
level of the GSTP1 enzyme. (Zimniak P. et al. (1994) Eur. J.
Biochem. 224:893-899). Recently it has been found that the 105Val
allele variant of the GSTP1 gene at exon 5 is associated with a low
GST enzyme activity in normal lung tissue and esophageal Barrett's
epithelium. (Watson M. A. et al. (1998) Carcinogenesis 19:275-280
and Van Leishout, E. M. M. (1999) Cancer Res. 59:588-589).
Additionally, it has been shown that the 105Val allele is
associated with increased risk for testicular, bladder cancer and
esophageal carcinoma, but not for colon or breast cancer. (Harries
L. W. (1997) Carcinogenesis 18:641-644).
[0150] Furthermore, this Ile105Val substitution has been shown to
be associated with better survival in women with breast cancer who
received chemotherapy (cyclophosphamide, 5-FU, adriamycin) (Sweeney
C. (2000) Cancer Res. 60:5621-5624). Nishimura et al. (Nishimura T.
et al. (1998) Chem. Biol. Interact. 111:187-198) showed that the
response rate of patients with head and neck cancer receiving
platinum-based chemotherapy was significantly higher for patients
with low GST protein expression. Based on these encouraging data 81
patients with advanced colorectal tumors that received combination
chemotherapy of 5-FU/oxaliplatin were genotyped.
[0151] Methods and Results: In this study, 81 patients with
advanced colorectal cancer, who received 5-FU/oxaliplatin
chemotherapy as a third line treatment, after failing 5-FU and
CPT-11 were screened for the polymorphism at exon 5 of the GSTP1
gene. The median overall survival time was 10.2 months (95% CI:
7.9, 13.3) with a median follow up time of 11 months (95% CI: 1.1,
15.3). Patients with a VAL/VAL genotype had a significant survival
benefit compared to patients heterozygous or homozygous for the ILE
allele (p=0.028, Log rank Test). Patients that are homozygous for
the VAL allele had a probability of survival at 18 months of 0.89,
compared to 0.40 for patients heterozygous and only 0.06 for
patients homozygous for the ILE allele. Patients homozygous for the
ILE allele showed a 5.4 fold increased relative risk of dying when
compared to the VAL/VAL group (Table 3). Patients homozygous for
the ILE allele had a median survival of 7.9 months (95% CI: 5.9,
12.8) compared to 13.3 months (95% CI: 8.4, 23.7) for heterozygous.
Patients homozygous far the VAL allele survived 24+ months (95% CI:
NA) (p=0.028, Log rank Test, Table 4).
TABLE-US-00003 TABLE 3 Univariate Analysis of Survival of Patients
with Colon Cancer Probability of No. Relative Survival at 18
Factors Patients Risk.sup.1 95% CI.sup.2 months p-Value.sup.3
GST-P1 0.028 VAL/VAL 9 1.00 0.89 .+-. 0.10 ILE/VAL 37 2.73 (1.43,
5.23) 0.40 .+-. 0.11 ILE/ILE 35 5.40 (2.83, 10.30) 0.06 .+-. 0.06
GST-P1 0.011 Any VAL 46 1.00 0.44 .+-. 0.11 ILE/ILE 35 2.12 (1.15,
4.18) 0.06 .+-. 0.06 GST-P1 0.14 VAL/VAL 9 1.00 0.89 .+-. 0.10 Any
ILE 72 3.93 (0.55, 28.23) 0.21 .+-. 0.07 .sup.1Relative risk can be
thought as the average increased chance of dying at any point in
time for patients in the second group compared to those In the
first group. The group with better prognosis is listed first.
.sup.295% confidence interval .sup.3Based on logrank test.
TABLE-US-00004 TABLE 4 Association between genotype of the GSTPI
gene and survival of patients with advanced colorectal cancer
Median Genotype No. of Patients Survial 95% CI.sup.1 p-value.sup.2
ILE/ILE 35 7.9 months (5.9, 12.8) 0.028 ILE/VAL 37 13.3 months
(8.4, 23.7) VAL/VAL 9 24+ months (NA) .sup.195% confidence interval
.sup.2Based on logrank test. Overall median time survival and its
95% CI: 10.2 (7.9, 13.3) months Overall median time follow up and
range: 11.0 (1.1, 25.3) months The survival far this study is not
related to GENDER and ETHNICITY.
[0152] Applicants show a significant association between survival
and the Ile105Val polymorphism at exon 5 of the GSTP1 gene.
Patients homozygous for the amino acid substitution had a
significant survival benefit. According to previous in-vitro
reports and studies in different human tissues the Val/Val genotype
is associated with a lower GST enzyme activity compared to the
heterozygous and the ILE/ILE genotype. (Zimniak P. et al. (1994)
supra; Watson M. A. et al., (1998) supra; and Van Lieshout (1999),
supra). Considering these results, patients with the VAL/VAL
genotype and respectively a lower GST enzyme activity benefit from
treatment with 5-FU/oxaliplatin compared to heterozygotes and the
ILE/ILE genotype group. A to GST enzyme activity is thought to be
less efficient in glutathione conjugation of drug intermediates,
which leads to a longer and most likely more efficient exposure of
the active drug to the tumor cell. This might explain the survival
benefit for patients with two or at least one VAL allele compared
to the ILE/ILE genotype.
[0153] To Applicants' knowledge, this is the first report of the
role of the ILE105VAL polymorphism of the GSTP1 gene and patients
with metastatic colorectal cancer, which received 5-FU/oxaliplatin
chemotherapy. This GSTP1 polymorphism can be become a useful marker
to identify patients with an increased risk to fail this third-line
chemotherapy, thus sparing those heavily pretreated patients the
side effects of a 5-FU/oxaliplatin therapy and refer them to other
therapy alternatives. The procedure of a simple blood test may be
enable the clinician to design more individualized
chemotherapy.
Example 8
A Polymorphic Dinucleotide Repeat in Intron 1 of EGFR Gene is
Associated with Clinical Response to Platinum Based Chemotherapy in
Patients with Advanced Colorectal Disease
[0154] EGFR is a 170-kD transmembrane glycoprotein whose gene is
located on the short arm of human chromosome 7p12. It is a member
of the receptor protein tyrosine kinase family with several
extracellular growth factor ligands, including epidermal growth
factor (EGF), and TGF-.alpha.. EGFR are frequently over expressed
in many types of human cancers, including CRC (colon and rectal
cancers) and their over expression typically confers a more
aggressive clinical behavior. (Salomon, D. et al. (1995) Critical
Reviews in Oncology-Hematology 19:183-232).
[0155] The level of EGFR expression is primarily regulated by the
abundance of its mRNA and the nature of the EGFR over expression is
believed to be due to an increase in the rate of EGFR
transcription. (Grandis, J. R. and Tweardy, D. J. (1993) Cancer
Res. 533579-3584). Recently, study shows that EGFR gene
transcription activity declines with increasing numbers of a highly
polymorphic dinucleotide repeat (CA repeat) in Intron 1 (Gebhardt
F. et al. (1999) J. Bio. Chem. 274:13176-13180).
[0156] The EGFR polymorphic dinucleotide repeat (CA repeat) of 78
patients to with metastatic colorectal cancer who previously had
failed 5-FU based chemotherapy was assessed to determine their
response and overall survival to 5-FU/oxaliplatin combination
treatment. The number of CA repeats was determinated by 5'-end
labeled polymerase chain reaction using forward primer
5'-GTTTGAAGAATTTGAGCCAAAC-C 3' (SEQ ID NO. 1) and reverse primer:
5'-TTCTTCTGCACACTTGGCAC 3' (SEQ ID NO. 2). The reaction was
incubated for 28 cycles with denaturation at 94.degree. C. for 1
minute, annealing at 55.degree. C. for 1 minute, and extension at
72.degree. C. for 2 minutes. The reaction products were separated
on 6% denaturing polyacrylamide DNA sequencing gels, vacuum blotted
and exposed to Kodak XAR film overnight using well known procedures
as described in Chi, D. D. et al. (1992) Human Molecular Genetics
1:135.
[0157] Thirty-eight patients were evaluated for response. The
overall response rate was 18% (7/38). 56% (4/9) patients were found
with the 16/16 repeats progressed, compared to 6% (1/17) and 8%
(1/12) of those with 16/18 repeats and 16/20 repeats respectively
(p=0.008 Fisher's exact test, two-tailed). The overall survival and
its relation to the polymorphism were also evaluated. For patients
with the 16/16 repeats the median survival was 66 days. Those with
the 16/18 repeats had a median survival of 179 days. Finally, those
with the 16/20 repeats had a median survival of 805 days (p=0.1 for
trend).
[0158] This report shows that short CA repeats (16/16) increase the
EGFR gene transcription and overexpressed the gene. Thus short CA
repeats (16/16) have a poor prognosis compare with long CA repeats
(16/20).
Example 9
Epidermal Growth Factor Receptor (EGFR) Gene Expression and
Polymorphism Predict Pelvic Recurrence in Patients with Rectal
Cancer Treated with Chemoradiation
[0159] EGFR is frequently overexpressed or mutated in many types of
cancer including colorectal cancer. EGFR overexpression is
associated with more aggressive tumor behavior and poor tumor
response to cytotoxic agents and radiation. In vitro and clinical
studies have associated EGFR overexpression with radioresistance.
Example 8, above, shows that a dinucletide repeat length
polymorphism in intron 1 of the EGFR gene was associated with
response to 5-FU/oxaliplatin in patients with metastatic colorectal
cancer. In vitro data also suggest that this genomic polymorphism
is associated with expression levels of EGFR.
[0160] Methods: Seventy-three patients with locally advanced rectal
cancer (UICC stage II and III) were treated with adjuvant
radio-chemotherapy. There were 25 (34.2%) women and 48 (65.8%) men
with a median age of 52 years (range 25, 79 years). Thirty-one
patients (42.5%) developed local tumor recurrence during the follow
up time. The tumors were graded histopathologically as highly
differentiated (G1; 1 patient) moderately differentiated (G2; 56
patients), and poorly differentiated (G3; 16 patients).
Histological staging revealed 22 patients stage T2, 51 patients
stage T3. Thirty-five (35) patients were lymph node negative, 38
had lymph node metastases. No patient had systemic metastases at
the time of first diagnosis. Ethnic background: 50 patients
Caucasian, 13 Hispanic, 8 Asian, 2 African-American. Patient data
were collected retrospectively. Informed consent was signed by all
patients involved in the study. Patients underwent lower anteriorer
resectomy (LAR; n=44), abdominal perineal resectomy (APR; n=20), or
transanal resectomy (TR; n=9), followed by 5-FU infusion plus
pelvic radiation. Pelvic irradiation was given as a dose of 45 Gy
to the whole pelvis and an additional boost up to 54 Gy.
[0161] Samples for gene expression analysis were obtained during
the surgical procedure. All samples were formalin-fixed and
paraffin-embedded. All paraffin embedded specimens underwent
laser-capture-microdissection in order to isolate RNA from tumor
tissue and adjacent normal tissue. RNA isolation after dissection
was done according to U.S. Pat. No. 6,248,535. Following RNA
isolation, cDNA was prepared from each sample. Quatification of
cDNA and an internal reference gene (beta [.beta.]-actin) was
conducted using a fluorescence-based real-time detection method
(ABI PRISM 7900 Sequence Detection System [TaqMan.RTM.];
Perkin-Elmer Applied Biosystems, Foster City, Calif.). The PCR
mixture consisted of 600 nmol/L of each primer, 200 nmol/L probe
(sequences used are given below), 5 units of AmpliTaq.RTM. Gold
polymerase, 200 .mu.mol/L each of dATP, dCTP, and dGTP, 400
.mu.mol/L dTTP, 3.5 mmol/L MgCl2, and 1.times. TaqMan.RTM. buffer A
containing a reference dye, to a final volume of 20 .mu.L (all
reagents were supplied by Perkin-Elmer Applied Biosystems). Cycling
conditions were 50.degree. C. for 10 seconds and 95.degree. C. for
10 minutes, followed by 46 cycles at 95.degree. C. for 15 seconds
and 60.degree. C. for 1 minute. Colon, liver, and lung RNAs (all
Stratagene, La Jolla, Calif.) were used as control calibrators on
each plate. Blood sample was collected from each individual and
genomic DNA was extracted from peripheral blood lymphocytes using
the QiaAmp kit (Qiagen, Valencia, Calif.). EGFR microsatellite
analysis was done according to well known procedures. Briefly,
standard PCR reactions were performed with 5'-.sup.33P-.gamma.ATP
end-labeled forward primer according to well known methods and the
reaction products were separated on 6% denaturing polyacrylamide
DNA sequencing gel, vacuum blotted and exposed to XAR film
(Eastman-Kodak Co., Rochester N.Y.) for overnight. Exact number of
EGFR CA repeat was confirmed by direct sequence the PCR
product.
[0162] Conclusion: A significant association between pelvic
recurrence and EGFR gene expression levels was found in rectal
normal tissues. Patients with high EGFR gene expression levels had
a higher (3.8 fold) relative risk for pelvic recurrence compared
with those have low EGFR gene expression levels (p=0.022 log rank
test) treated with chemoradiation. A trend of possible relationship
(P=0.17 log rank test) exists between dinucleotide repeat length
polymorphism in intron 1 of the EGFR gene and time to local
recurrence in rectal cancer patients. Patients with shorter (CA)n
repeat (both CA repeat<20) have a shorter time to local
recurrence compared with those with longer (CA)n repeat (both CA
repeat.gtoreq.20). EGFR gene expression levels in tumor tissue do
not have statistical significance to predict time to local
recurrence in rectal patients treated with chemoradiation.
[0163] This is the first study that shows EGFR gene expression
levels in normal rectal tissue and the dinucleotide repeat length
polymorphism in intron 1 of the EGFR gene may be associated with
time to local recurrence in rectal cancer patients treated with
chemoradiation. These data suggest that EGFR gene expression levels
in normal rectal tissues and EGFR gene polymorphism can identify
patients at high risk for pelvic recurrence.
Example 10
COX-2 Prevents Clinical Toxicity Associated with Chemotherapy
[0164] Prostaglandines (PG) are formed by the action cyclooxygenase
(COX). Two related isoforms, COX-1 and COX-2 transform arachidonic
acid to prostaglandins, but they differ in their physiological
roles and distribution (Smith W. L. et al. (1996) J Biol Chem.
271:33157-33160). COX-1, a constitutive isoform is present in many
cell types throughout the human body and is specifically
responsible for the maintaining of gastric mucosa by production of
cytoprotective prostanoids, appropriate platelet function, and
renal blood flow.
[0165] The inducible isoform COX-2, has been shown to be absent
under normal conditions, but to be induced by cytokines, growth
factor, mitogenes and tumor promotors and to be responsible for
mediation of inflammation, fever, tumor growth and pain. (Hla T.
and Neilson K. (1992) Proc Natl Acad Sci USA 89:7384-7388 and Jones
D. A. et al. (1993) J. Biol. Chem. 268:9049-9054).
[0166] Classic NSAIDs inhibit both isoforms at standard
anti-inflammatory doses. The inhibition of COX-2 explains the
therapeutic effects since it is involved in the formation of PG
that mediates pain and inflammation. But unwanted side effects such
as gastric toxicity, mild bleeding diathesis, and renal dysfunction
also occur, because of the concurrent inhibition of COX-1.
(Dannhardt G. and Kiefer W (2001) Eur. J. Med. Chem. 36:109-126).
Recently, selective inhibitors of cyclooxygenase-2, have been
proven to exert therapeutic efficacy without these unwanted side
effects (Reddy B. S. et al. (1996) Cancer Res. 56:4566-4569).
[0167] Since cancer patients, especially in advanced stages of the
disease, often experience tremendous pain, drugs are needed that
provide effective relief But it has to be considered that side
effects from these painkillers may interfere with the toxicity that
is caused by the necessary chemotherapy. Since the prostaglandin
pathway plays a significant role in secretory diarrhea, inhibition
of COX-2 may in fact decrease diarrhea caused by chemotherapy.
(Beubler E. and Schuligoi R. (2000) Annals of the New York Academy
of Sciences. 915:339-46). Animal data suggest that COX-2 inhibition
in fact can inhibit or prevent chemically induced neurotoxicity in
brain of rats suggesting a potential role of COX-2 in
neurotoxicity. (Kunz T. and Oliw E. H. (2001) Eur. J. of
Neuroscience 13(3):569-75 and Hewett S. J. et al. (2000) J.
Pharmacology & Experimental Therapeutics 293(2):41 7-25).
[0168] Selective COX-2 inhibitors will be most beneficial
symptomatic treatment for the patient, who receives chemotherapy
for two reasons. First, the patient's pain will be reduced since
COX-2 inhibitors block the formation of pain and inflammation
mediating prostaglandins. Second, the formation of cytoprotective
prostaglandins in the gastrointestinal tract will not be altered.
The physiological protection against aggressive compounds such as
acid and agents from chemotherapy regimens will be kept. This may
result in less side effects from chemotherapy. Additionally, it has
been suggested that the rate-limiting enzyme in the PG pathway,
COX-2, which is highly expressed in many tumors (Bae S. H. et al.
(2001) Clin. Cancer Res. 7:1410-1418) is associated with the
carcinogenesis process in colorectal cancer (Sano H. et al., Cancer
Res. 55: 3785-3789 and Hao X. (1999) et al. (1999) J. Pathol.
187:295-301). The induction of colorectal tumors by azzoxymethane
has been shown to be nearly complete suppressed by selective COX-2
inhibition (Kawamori T. et al. (1998) Cancer Res. 58:409-412) and
colon polyps showed regression after treatment with non-selective
NSAIDs (Giardiello F. M. et al. (1993) N. Engl. J. Med.
328:1313-1316).
[0169] In a retrospective analysis, Applicants have identified that
celebrex at doses between 200-400 mg day is protecting against
neurotoxicity in patients treated with oxaliplatin. In the analysis
of 156 patients, 90 of which had no celebrex treatment in
combination with oxaliplatin and 5-FU, 26 of these patients
developed grade II or III neurotoxicity, from the 56 patients who
had celebrex therapy in combination with oxaliplating/5-FU
chemotherapy, only 3 developed grade II or lit neurotoxicity. This
is highly statistically significant (p<0.01).
Example 11
Functional Polymorphisms of Matrix Metalloproteinases can Predict
Distant Metastases in Patients with Advanced Colorectal Cancer
[0170] Matrix metalloproteinases (MMPs) are members of a family of
zinc-dependent enzymes involved in the degradation of extracellular
matrix (ECM). In vitro studies have shown that the MMPs are able to
degrade an array of connective tissue proteins, suggesting that
these enzymes may play a role in connective tissue destruction and
formation associated with various pathological processes including
cancer invasion and metastasis, cartilage destruction in arthritis,
atherosclerotic plaque rupture, and the onset of aneurysms.
[0171] Naturally occurring sequence variation has been discovered
in the promoter regions of a number of MMP genes, including MMP-1
(-1607 1G/2G), MMP-3 (-1612 5A/6A), and MMP-9 (-1562 C/T). These
germline polymorphisms have been shown to have allele-specific
effects on the transcriptional activities of these MMP gene
promoters.
[0172] In fact, ovarian tumor tissues from patients possessing the
2G allele within the promoter of the MMP-1 (collagenase-1) gene
have been shown to express more MMP-1 compared to those from
patients not carrying the 2G allele. (Kanamori Y. et al. (1999)
Cancer Research 59:4225-4227). Insufficient MMP-3 (stromelysin-1)
expression has been attributed to the presence of 6A allele within
the gene promoter leading to vascular matrix, and a study has
demonstrated that the 6A/6A genotype is associated with increased
carotid artery wall thickness measured using non-invasive
ultrasonography. (Gnasso A. et al. (2000) Arterioscler. Thromb.
Vase. Biology 20:1600-1605). In a cohort study of Caucasian
patients with coronary atherosclerosis, a correlation of the
C-1562T polymorphism of MMP-9 (gelatinase B) gene with severity of
the disease has been identified, and this association may be due to
enhanced ability of vascular smooth muscle cells to migrate and
proliferate during atherogenesis in individuals possessing the T
allele. (Zhang B et al. (1999) Circulation 99:1788-1794).
[0173] Methods: From 1998 through 2000, 472 patients with advanced
colorectal cancer who were treated at University of Southern
California/Norris Comprehensive Cancer Center were identified. Of
these 472 patients, the association between the polymorphisms of
MMP-1, MMP-3, and MMP-9 and site of metastases in 60 participants
who were eligible for the analysis of present study were examined.
This study was investigated at the Norris comprehensive Cancer
Center and approved by the Institutional Review Board (IRB) of the
University of Southern California for Medical Sciences. The age,
ethnicity, and follow-up information for each subject were obtained
from the retrospective chart reviews.
[0174] A blood sample was collected from each patient and the
corresponding genomic DNA was extracted from the peripheral blood
lymphocytes using the QiaAmp kit (Qiagen, Valencia, Calif., USA).
All samples were evaluated using a PCR-RFLP technique. After
restriction enzyme digestion, PCR products were visualized on a 4%
agarose gel and analyzed.
[0175] Results For the study participants, 10 patients only had
peritoneal carcinomatosis with a median follow-up of 17 months (95%
CI, 10.6-25.3 months) before tumor progression; and 50 patients who
presented liver and/or lung metastases had a median follow-up of
34.3 months (95% CI, 11.6-61.3 months) before tumor progression. Of
these patients, the median age was 55.3 years (36.2-91.1 years) and
the median follow-up was 30.1 months (95% CI, 10.6-61.3
months).
[0176] The analyses of MMP-3 and MMP-9 polymorphisms failed to show
statistical significance (p=0.18 and p=0.69, respectively).
However, the presence of 2G allele, which has been implicated in
higher transcription rate of MMP-1 gene, was associated with site
of metastases when patients with peritoneal disease were compared
to those with distant metastases (p=0.08).
[0177] Fourteen patients (23%) possessed the MMP-1 1G/1G genotype,
15 patients (25%) had 2G/2G genotype, and 30 patients (50%) were
heterozygous for this variant. Of the study participants with only
peritoneal carcinomatosis, 40% (4/10) had the 1G/1G genotype, while
0% (0/10) of those with 2G/2G genotype and 60% (6/10) of those
heterozygous showed evidence of local disease. Of the patients who
had distant metastases, 20% (10/49) carried the homozygous 1G
allele compared to (15/49) with homozygous 2G allele and 49%
(24/49) with heterozygous genotype.
[0178] The 2G allele has been associated with deep invasive primary
tumors, and therefore, poorer prognosis in patients with cutaneous
malignant melanoma (CMM), suggesting that the aggressiveness of CMM
is influenced by variation in the MMP-1 gene promoter. (Ye S. et
al. (2001) Cancer Research 61:1296-1298). MMP-1 expression has been
implicated as a novel marker for hematogenous metastasis of
colorectal cancer, implying that its inhibition may be a strategy
for prevention of metastasis. (Sunami E. et al. (2000) The
Oncologist 5:108-114). MMP-1 immunoreactivity has also been
significantly correlated with lymph node and hepatic metastases,
tumor growth pattern, and additionally with the presence of
lymphatic, venous, and neural invasions. (Shiozawa J. et al. The
U.S. and Canadian Academy of Pathology 13(9):925-933).
[0179] Thus, the inheritance of the 2G allele is shown to be
associated with invasiveness of colorectal cancer and distant
metastases. Particularly, patients carrying homozygous 2G allele
can be more genetically susceptible to developing distant
metastases due to increased degradation of ECM, facilitating
angiogenesis.
Example 12
Association Between Genetic Polymorphisms of Interleukin-8(IL-8)
and its Receptor CXCR1 and Survival of Patients with Metastatic
Colorectal Cancer Treated with 5FU/Oxaliplatin
[0180] Interleukin 8(IL-8) a member of the CXC chemokine family is
known to be involved in tumor cell growth and metastasis in
colorectal cancer. (Xie K. (2001) Cytokine Growth Factor Rev
12(4):375-91.) Its receptors CXCR1 and CXCR2 have vital roles in
tumor progression and angiogenesis. (Miller L. J. et al. (1998)
Anticancer Res. 18(1A):77-81). Studies show that expression of
IL-8, CXCR1, and CXCR2 contribute to tumor progression and
metastases in vitro and in vivo. (Brew R. et al. (2000) Cytokine
12(1):78-85) and Li A. et al. (2001) Clin. Cancer Res.
7(10):3298-304). Polymorphisms in the promoter region of IL-8 gene
(T-251A) and a novel polymorphism in exon 2 of CXCR1 gene
(Ser+2607Thr) may influence the expression of IL-8 and its receptor
and therefore influence clinical outcome of patients with
metastatic colorectal cancer. (Hull J. A. et al. (2000) Thorax
55(12):1023-7 and Renzoni E. et al. (2000) Arthritis Rheum
43(7):1633-40) The hypothesis that patients with genomic
polymorphisms associated with higher expression or activity of
Interleukin would have poorer prognosis was tested.
[0181] Patients were enrolled in the compassionate oxaliplatin
protocol 3C-98-3 at the University of Southern California/Norris
Comprehensive Cancer Center from 1998-2000. The chemotherapeutic
regimen was as follows: 130 mg/m2 oxaliplatin every three weeks and
continuous infusion 5-FU (200 mg/m2/d). All patients had failed a
prior treatment with 5-FU and 79% failed an additional second line
treatment with irinotecan (CPT-11). Survival was determined from
the start day of the 5-FU/oxaliplatin chemotherapy to death. Time
to progression was determined from the start day of chemotherapy to
the day the patient was taken off study due to disease progression.
Patients who were alive at the last follow-up evaluation were
censored at that time. Responders to therapy were classified as
those patients whose tumor burden decreased by 50% or more for at
least six weeks. Progressive disease was defined as 25% or more
increase in tumor burden or the appearance of new lesions. Patients
who did not experience a response and did not progress within the
first 12 weeks following start of 5-FU/oxaliplatin, were classified
as having stable disease. Four patients dropped out of the study
too early for evaluation of response but they were included for the
determination of survival.
[0182] A blood sample was collected from each individual and
genomic DNA was extracted from peripheral blood lymphocytes using
the QiaAmp kit (Qiagen, Valencia, Calif.). IL-8 and CXCR1
polymorphisms were done using PCR-RFLP.
[0183] Results: The overall response rate in patient groups was 9%.
Only 16 patients remain alive and the follow-up from 6 to 18
months. Median survival was 9.4 months (95% C.I. 7.6-12.8) and
median time to progression was 5.0 months (95% C.I. 4.4-6.5).
[0184] A significant relationship (P<0.05) was found between
CXCR1 exon 2 Ser +2607 Thr polymorphism and the overall survival of
patients with colorectal cancer treated with 5-FU/oxaliplatin after
stratified by ECOG, histology or prior CPT-11 treatment.
[0185] Patients with the CXCR1GC genotype had the 2.35 fold of
relative risk of dying compared to patients with GG genotype after
stratified by ECOG, histology or prior CPT-11 treatment. There is
no significant relationship between IL-8 promoter polymorphism and
overall survival. Also, there is no significant relationship
between clinical response and both polymorphisms.
[0186] This is the first time study shows the IL-8 receptor CXCR1
Ser +2607 Thr polymorphism can be associated with overall survival
in colorectal cancer patients treated with platinum-based
chemotherapy.
[0187] The preceding examples are intended to illustrate, but not
limit, the inventions as described herein.
Sequence CWU 1
1
2123DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 1gtttgaagaa tttgagccaa acc 23220DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
2ttcttctgca cacttggcac 20
* * * * *