U.S. patent application number 10/917330 was filed with the patent office on 2005-05-05 for method for detecting cell proliferative disorders.
This patent application is currently assigned to The Johns Hopkins University School of Medical. Invention is credited to Sidransky, David.
Application Number | 20050095621 10/917330 |
Document ID | / |
Family ID | 21828150 |
Filed Date | 2005-05-05 |
United States Patent
Application |
20050095621 |
Kind Code |
A1 |
Sidransky, David |
May 5, 2005 |
Method for detecting cell proliferative disorders
Abstract
The present invention relates to the detection of a cell
proliferative disorder associated with alterations of
microsatellite DNA in a sample. The microsatellite DNA can be
contained within any of a variety of samples, such as urine,
sputum, bile, stool, cervical tissue, saliva, tears, or cerebral
spinal fluid. The invention is a method to detect an allelic
imbalance by assaying microsatellite DNA. Allelic imbalance is
detected by observing an abnormality in an allele, such as an
increase or decrease in microsatellite DNA which is at or
corresponds to an allele. An increase can be detected as the
appearance of a new allele. In practicing the invention, DNA
amplification methods, particularly polymerase chain reactions, are
useful for amplifying the DNA. DNA analysis methods can be used to
detect such a decrease or increase. The invention is also a method
to detect genetic instability of microsatellite DNA. Genetic
instability is detected by observing an amplification or deletion
of the small, tandem repeat DNA sequences in the microsatellite DNA
which is at or corresponds to an allele. The invention is also a
kit for practicing these methods.
Inventors: |
Sidransky, David;
(Baltimore, MD) |
Correspondence
Address: |
BANNER & WITCOFF
1001 G STREET N W
SUITE 1100
WASHINGTON
DC
20001
US
|
Assignee: |
The Johns Hopkins University School
of Medical
Baltimore
MD
|
Family ID: |
21828150 |
Appl. No.: |
10/917330 |
Filed: |
August 13, 2004 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10917330 |
Aug 13, 2004 |
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09948909 |
Sep 10, 2001 |
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6780592 |
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09948909 |
Sep 10, 2001 |
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08968733 |
Aug 28, 1997 |
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6291163 |
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60025805 |
Aug 28, 1996 |
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Current U.S.
Class: |
435/6.12 |
Current CPC
Class: |
C12Q 2600/112 20130101;
C12Q 2600/158 20130101; C12Q 1/6886 20130101; C12Q 2600/156
20130101; C12Q 2600/118 20130101; C12Q 2600/172 20130101; C12Q
1/6883 20130101 |
Class at
Publication: |
435/006 |
International
Class: |
C12Q 001/68 |
Claims
1-29. (canceled)
30. A method for detecting cancer or precancer in a subject, the
method comprising: detecting in test sample DNA of the subject an
allelic imbalance at a genetic locus by determining and comparing
level of microsatellite DNA present at a first allele to level of
microsatellite DNA present at a second allele, wherein the subject
is heterozygous for the genetic locus, wherein the first and second
alleles are at the genetic locus and the genetic locus comprises
microsatellite DNA, wherein the test sample DNA is from a cell of
an organ which drains into the test sample, wherein the test sample
is selected from the group consisting of the subject's:
lymphocytes, cerebral spinal fluid, and cervical tissue, wherein
detection of an allelic imbalance is indicative of cancer or
precancer.
31. The method of claim 30 wherein the allelic imbalance is a
decrease in the level of microsatellite DNA present at the first
allele.
32. The method of claim 31 wherein the level of microsatellite DNA
present at the first allele in the test sample DNA is less than 50%
of the level of microsatellite DNA present at the first allele in a
control sample of the subject wherein the control sample lacks
cancerous or precancerous cells.
33. The method of claim 30 wherein the step of detecting comprises
size fractionation of the first and second alleles.
34. The method of claim 33 wherein the first and second alleles are
fractionated by gel electrophoresis.
35. The method of claim 30 wherein the subject has cancer.
36. The method of claim 30 wherein the microsatellite DNA is
amplified prior to the step of detecting.
37. The method of claim 36 wherein said step of amplifying
comprises a polymerase chain reaction.
38. The method of claim 30 wherein the genetic locus is selected
from the group consisting of: DRPLA, UT762, IFNA, D9S200, D9S156,
D3S1284, D3S1238, CHRNB1, D17S86, D9S747, D9S171, D16S476, D4S243,
D14S50, D21S1245, FgA, D8S3G7, THOO, D115488, D135802, D175695,
D175654, and D20548.
39. The method of claim 30 wherein said step of amplifying is
performed using primers that hybridize to nucleotide sequences
selected from the group consisting of SEQ ID NO: 1-31 and SEQ ID
NO:32.
40. The method of claim 39 wherein said primers are selected from
the group consisting of SEQ ID NO: 33-63 and SEQ ID NO:64.
41. The method of claim 30, wherein the cancer or precancer is not
due to a DNA repair gene defect.
42. The method of claim 41 wherein the subject has cancer.
43. The method of claim 42 wherein the subject has a benign
neoplasm.
44. The method of claim 42 wherein the subject has a malignant
neoplasm.
48. A method for detecting cancer or precancer in a subject, the
method comprising: detecting in test sample DNA of the subject an
allelic imbalance at a genetic locus by determining and comparing
level of microsatellite DNA present at a first allele to level of
microsatellite DNA present at a second allele, wherein the subject
is heterozygous for the genetic locus, wherein the first and second
alleles are at the genetic locus and the genetic locus comprises
microsatellite DNA, wherein the test sample DNA is from a cell of
an organ which drains into the test sample, wherein the test sample
is selected from the group consisting of the subject's: urine,
sputum, bile, stool, saliva, tears, serum, and plasma, wherein
detection of an allelic imbalance is indicative of cancer or
precancer.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] The present invention claims priority under 35 U.S.C.
.sctn.119(e) from Provisional Application Ser. No. 60/025,805,
filed Aug. 28, 1996.
BACKGROUND OF THE INVENTION
[0002] 1. Field of the Invention
[0003] The present invention relates generally to the detection of
a target nucleic acid sequence and specifically to the detection of
microsatellite DNA sequence mutations associated with a cell
proliferative disorder.
[0004] 2. Description of Related Art
[0005] Cancer remains a major cause of mortality worldwide. Despite
advancements in diagnosis and treatment, the overall survival rate
has not improved significantly in the past twenty years. There
remains an unfulfilled need for a more sensitive means of early
diagnosis of tumors, before the cancer progresses.
[0006] One of the most serious cancers is bladder cancer. Bladder
cancer is the fourth most common cancer in men and the eighth most
common in women. Transitional cell carcinoma (TCC) of the bladder
is the most common urothelial malignancy of the urinary tract, with
an incidence of approximately 51,000 each year in the United States
alone.
[0007] One reason that bladder cancer is so serious is because,
presently, detecting and treating bladder cancer is difficult.
Seventy percent of patients with an initial diagnosis of
transitional cell carcinoma have superficial tumors, which can be
treated by transurethral resection alone. Approximately 70% of
these patients continue to suffer from recurrent disease, and 15%
develop lesions that invade muscle within the first two years.
[0008] Detecting tumor recurrence in patients with transitional
cell carcinoma of the bladder requires close surveillance. Urine
cytology is a common non-invasive procedure for the diagnosis of
this disease, but it can miss up to 50% of tumors. The "gold
standard" for diagnosis is cystoscopy, which allows visualization
and direct biopsies of suspicious bladder lesions in the mucosa.
However, because cystoscopy is an expensive and invasive procedure,
it cannot be used as a general screening tool for the detection of
bladder cancer.
[0009] Other serious cancers are the head and neck cancers. Head
and neck cancer remains a morbid and often fatal disease. Large
tumor bulk and tumor extension are predictors of a local regional
recurrence and poor outcome. Detection of occult neoplastic cells
in surrounding surgical margins is a strong predictor of local
regional recurrence resulting in a significant decrease in overall
survival.
[0010] DNA contains unique sequences interspersed with moderately
and highly repetitive DNA sequences. Variations in the repetitive
sequence elements such as minisatellite (or variable number tandem
repeat) DNA sequences and microsatellite (or variable simple
sequence repeat) DNA sequences have been useful for chromosomal
identification, primary gene mapping, and linkage analysis.
Microsatellite DNA sequences are an especially common and highly
polymorphic class of genomic elements in the human genome. One
advantage to the use of repetitive sequence variations is the
greater number of alleles present in populations compared with
unique genetic sequence variations. Another advantage is the
ability to readily detect sequence length variations using the
polymerase chain reaction for the rapid and inexpensive analysis of
many DNA samples.
[0011] Tumors progress through a series of genetic mutations. These
genetic mutations can be used as specific markers for the detection
of cancer. One set of genetic mutations that can be used to detect
the presence of cancer is the loss of chromosomes. Diploid
organisms, including humans, have pair of chromosomes for each
member of the chromosomal set. Tumor cells will characteristically
lose chromosomes, resulting in a single chromosome, rather than a
pair of chromosomes, for each member of the chromosomal set.
Chromosomal deletions and additions are an integral part of
neoplastic progression and have been described in most kinds of
cancers. A pair of chromosomes has two alleles for a genetic locus
is heterozygous for that locus; therefore, the heterozygosity
correlates to the cell having a pair of chromosomes. For years,
these chromosomal deletions or amplifications were detected through
the loss of heterozygosity.
[0012] Another of the genetic mutations used to detect the presence
of cancer is genetic instability. Genetic recombination tends to
occur most frequently at regions of the chromosome where the DNA is
homologous (where the DNA has a high degree of sequence
similarity). Where a DNA sequence is repetitive, the DNA homology
is greater. The DNA homology occurs not only at the same genetic
locus on the other pair of chromosomes, but also on other genetic
loci or within the same locus on the same chromosome. Normal
(non-tumor) cells tend to suppress this genetic recombination.
Tumor cells, however, characteristically undergo increased genetic
recombination. Where a DNA sequence is repetitive, genetic
recombination can result in the loss of repeat DNA sequences or the
gain of repeat DNA sequences at a genetic locus.
[0013] Microsatellite DNA instability has been described in human
cancers. Microsatellite DNA instability is an important feature of
tumors from hereditary non-polyposis colorectal carcinoma patients
(Peltomki et al., Science, 260: 810 (1993); Aaltonen et al.,
Science, 260: 812 (1993); Thibodeau et al., Science, 260: 816
(1993)). Microsatellite DNA instability by expansion or deletion of
repeat elements has also been reported in colorectal, endometrial,
breast, gastric, pancreatic, and bladder neoplastic tissues
(Risinger et al., Cancer Res., 53: 5100 (1993); Had et al., Cancer
Res., 53: 5087 (1993); Peltomki et al., Cancer Res. 53: 5853
(1993); Gonzalez-Zulueta et al., Cancer Res. 53: 5620 (1993)).
[0014] Some methods have been developed to detect the multiple
genetic changes that occur during the development of primary
bladder cancer. For example, mutations in the tumor suppressor gene
p53 signal the progression to invasiveness and have been
successfully used as molecular markers to detect cancer cells in
urine samples. However, this diagnostic strategy has limited
clinical application because the techniques are cumbersome and
because p53 mutations appear relatively late in the disease.
[0015] Because early diagnosis of bladder cancer is critical for
successful treatment, there is a pressing need for more sensitive
and cost-effective diagnostic tools. Both patients and physicians
would benefit from the development of improved non-invasive methods
for cancer surveillance.
SUMMARY OF THE INVENTION
[0016] The present invention provides a fast, reliable, sensitive
and non-invasive screening method for the detection of a cell
proliferative disorder in a subject. The method detects an allelic
imbalance by assaying microsatellite DNA, wherein an abnormality in
an allele is indicative of an allelic imbalance. Such abnormalities
include an increase or decrease in microsatellite DNA that is at or
corresponds to an allele. A decrease can be detected such that the
level of DNA corresponding to the allele is less than 50% of the
level of DNA of a corresponding allele in a microsatellite DNA
sample of a subject that lacks the cell proliferative disorder. An
increase can be detected as the appearance of a new allele.
[0017] The cell proliferative disorder detected by the method of
the invention may be a neoplasm, for example, a neoplasm of the
head, neck, lung, esophageal, stomach, small bowel, colon, bladder,
kidney, or cervical tissue. The sample of microsatellite DNA may be
urine, sputum, bile, stool, cervical tissue, saliva, tears,
cerebral spinal fluid, serum, plasma, or lymphocytes, for
example.
[0018] The microsatellite DNA detected by the method may be a locus
such as DRPLA, UT762, IFNA, D9S200, D9S156, D3S1284, D3S1238,
CHRNB1, D17S86, D9S747, D9S171, D16S476, D4S243, D14S50, D21S1245,
FgA, D8S3G7, THO, D115488, D135802, D175695, D175654, and
D20548.
[0019] The invention also provides a fast, reliable, sensitive and
non-invasive screening method for detecting genetic instability of
microsatellite DNA. An amplification or deletion of the small
tandem repeat DNA sequences indicates genetic instability in the
microsatellite DNA that is at or corresponds to an allele.
[0020] The present invention also provides a kit for detecting a
cell proliferative disorder, comprising oligonucleotide primers
that are complementary to a nucleotide sequence that flanks
nucleotide repeats of microsatellite DNA. In one embodiment, the
kit further comprises a detectably labeled deoxyribonucleotide.
BRIEF DESCRIPTION OF THE DRAWINGS
[0021] FIG. 1 is a table of patients with bladder lesions and urine
analysis (Cyto, cytology; LOH, loss of heterozygosity; Alt,
microsatellite).
[0022] FIG. 2 is a table of microsatellite analysis of urine
sediment.
[0023] FIG. 3 is a table of microsatellite analysis and clinical
outcome in head and neck cancer patients.
[0024] FIG. 4 is a table of microsatellite analysis of patients
with alterations in plasma.
[0025] FIG. 5 is a table of cytology and molecular status of
patients with bladder cancers.
DETAILED DESCRIPTION OF THE INVENTION
[0026] The present invention relates to a non-invasive method for
the detection of a cell proliferative disorder associated with
mutations of microsatellite DNA in a subject. The invention
provides a method for detecting an allelic imbalance by assaying
microsatellite DNA. The invention also provides a method for
detecting genetic instability of microsatellite DNA. The present
invention is based on the three following principles:
[0027] First, chromosomal deletions and genetic recombination are
an integral part of neoplastic progression and have been described
in most kinds of cancers. Allelic imbalance (loss of
heterozygosity) and genetic instability can now be detected in
clinical samples composed mostly of normal-looking (morphologically
normal) cells. The clinical samples can be readily obtained, thus
providing a non-invasive alternative to surgery and microdissection
of neoplastic tissue.
[0028] Second, monoclonality is a fundamental characteristic of
neoplasms. Clonal genetic mutations are integrally involved in the
progression of all cancers. Detection of a clonal population of
cells harboring a chromosomal deletion or amplification is
synonymous with the detection of cancer at a molecular level.
[0029] Third, microsatellite DNA in clinical samples can be
amplified in vitro to detect an allelic imbalance (loss of
heterozygosity) or genetic instability of microsatellite DNA. A
combination of markers for each tumor type can now be used to
identify many tumors in a given clinical sample. Moreover, these
markers can also be multiplexed in a single amplification reaction
to generate a low cost, reliable cancer screening test for many
cancers simultaneously.
[0030] In one embodiment, the invention provides a method for
detecting a cell proliferative disorder in a subject by detecting,
in a sample of microsatellite DNA from the subject, an allelic
imbalance. The presence of an allelic imbalance is indicative of a
cell proliferative disorder.
[0031] The term "cell-proliferative disorder" includes both benign
and malignant cell populations that morphologically differ from the
surrounding tissue. For example, the method is useful for detecting
tumors of the lung, breast, lymphoid, gastrointestinal, and
genitourinary tract; epithelial carcinomas that include
malignancies such as most colon cancers, renal-cell carcinoma,
prostate cancer, non-small cell carcinoma of the lung, cancer of
the small intestine, stomach cancer, kidney cancer, cervical
cancer, cancer of the esophagus, and any other organ type that has
a draining fluid or tissue accessible to analysis; and nonmalignant
cell-proliferative diseases such as colon adenomas, hyperplasia,
dysplasia and other pre-malignant lesions. Any disorder that is
etiologically linked to mutations in a microsatellite DNA locus is
susceptible to detection. In one embodiment, the method of the
invention is useful for the detection of transitional cell
carcinoma of the bladder and for the detection of head and neck
cancer.
[0032] A cell proliferative disorder as described herein may be a
neoplasm. Such neoplasms are either benign or malignant. The term
"neoplasm" refers to a new, abnormal growth of cells or a growth of
abnormal cells that reproduce faster than normal. A neoplasm
creates an unstructured mass (a tumor) which can be either benign
or malignant. For example, the neoplasm may be a head, neck, lung,
esophageal, stomach, small bowel, colon, bladder, kidney, or
cervical neoplasm. The term "benign" refers to a tumor that is
noncancerous, e.g. its cells do not proliferate or invade
surrounding tissues. The term "malignant" refers to a tumor that is
metastastic or no longer under normal cellular growth control.
[0033] The term "allelic imbalance" refers to the chromosomal loss
or gain that is characteristic of tumor cells. Diploid organisms,
including humans, have a pair of chromosomes for each member of the
chromosomal set. Tumor cells characteristically lose chromosomes,
often resulting in a single chromosome, rather than a pair of
chromosomes, for each member of a chromosomal set. Tumor cells also
on occasion gain chromosomes, resulting in a two or more
chromosomes, rather than a pair of chromosomes, for each member of
the chromosomal set.
[0034] When a genetic locus on the chromosome has a different DNA
sequence on each chromosome, a diploid organism has two alleles for
that genetic locus. A pair of chromosomes with two alleles for a
genetic locus is heterozygous. Whether a genetic locus is
heterozygous for a subject can readily be determined by analyzing a
sample of DNA from the normal (non-tumor) cells of the subject.
Because microsatellite DNA is polymorphic, a genetic locus that
contains microsatellite DNA will frequently be heterozygous. When a
tumor cell loses or gains a chromosome, the result is that the cell
loses or gains an additional copy of one of the alleles, causing an
allelic imbalance (loss of heterozygosity).
[0035] Microsatellite DNA markers that are heterozygous in normal
(non-tumor) cell DNA can be used to detect mutations in tumor cell
DNA. The loss of one allele identifies chromosomal deletions after
gel electrophoresis or other techniques. An imbalance between the
two alleles also identifies chromosomal amplifications. To do these
analyses by conventional methods requires extensive microdissection
of neoplastic cells so that normal (non-tumor) contaminating cells
would not disrupt the assay. The method of the invention, by
contrast, provides a non-invasive sampling technique in which the
presence of normal (non-tumor) cells does not interfere with the
assay. A loss of heterozygosity correlating with bladder cancer can
be detected in urine samples in patients where the samples contain
both normal and tumor cells. A loss of heterozygosity can be
detected in the plasma and saliva of patients with head and neck
cancer.
[0036] A combination of microsatellite DNA markers may be amplified
in a single amplification reaction. The markers are multiplexed in
a single amplification reaction, for example, by combining primers
for more than one locus. For example, DNA from a urine sample can
be amplified with three different randomly labeled primer sets,
such as those used for the amplification of the FgA, ACTBP2 and AR
loci, in the same amplification reaction. The reaction products are
separated on a denaturing polyacrylamide gel, for example, and then
exposed to film for visualization and analysis.
[0037] The term "microsatellite DNA" refers to mononucleotide,
dinucleotide, or trinucleotide sequences where alleles differ by
one or more repeat units. Microsatellite DNA is an especially
common and highly polymorphic class of genomic elements in the
human genoine. The microsatellite DNA most preferred in the method
of the invention has a sequence (X).sub.n, wherein X is the number
of nucleotides in the repeat sequence and is greater than or equal
to 1, preferably greater than or equal to 2, and most preferably
greater than or equal to 3 and wherein n is the number of repeats
and is greater than or equal to 2, and preferably from 4 to 6. When
X is 2, the nucleotide sequence may be TC. When X is 3, the
nucleotide sequence may be selected from AGC, TCC, CAG, CAA, and
CTG. Two examples of trinucleotide repeats are D1S50 and DRPLA
markers. Preferably when X is 4, the nucleotide sequence may be
selected from AAAG; AGAT and TCTT. Two examples of tetranucleotide
repeats are included in D21S1245 and FgA markers.
[0038] The microsatellite DNA sequence may be genetically linked to
a unique locus. For example, microsatellite DNA mutations may be
detected using a marker selected from ARA (chromosome X), D14S50
(chromosome 14), AR (chromosome X), MD (chromosome 19), SAT
(chromosome 6), DRPLA (chromosome 12), ACTBP2 (chromosome 6), FgA
(chromosome 4), D4S243 (chromosome 4), and UT762 (chromosome 21).
Tandem repeat sequences have been identified as associated with
Huntington's disease (HD), fragile X syndrome (FX), myotonic
dystrophy (MD), spinocerebellar ataxia type I (SCA1), spino-bulbar
muscular dystrophy, and hereditary dentatorubralpallidoluysian
atrophy (DRPLA).
[0039] The term "sample of microsatellite DNA" refers to DNA
present in or prepared from any tissue of a subject. The nucleic
acid from any specimen, in purified or nonpurified form, can be
used as the starting nucleic acid or acids, provided it contains,
or is suspected of containing, the specific nucleic acid sequence
containing the target nucleic acid. Thus, the process may employ,
for example, DNA or RNA, including messenger RNA (mRNA). The DNA or
RNA may be single stranded or double stranded. When RNA is used as
a template, enzymes and conditions optimal for reverse transcribing
the template to DNA would be used. A DNA-RNA hybrid that contains
one strand of each may also be used. A mixture of nucleic acids may
also be employed, or the nucleic acids produced in a previous
amplification reaction herein, using the same or different primers
may be so used. The mutant nucleotide sequence to be amplified may
be a fraction of a larger molecule or can be present initially as a
discrete molecule, such that the specific sequence is the entire
nucleic acid. It is not necessary that the sequence to be amplified
be present initially in a pure form; it may be a minor fraction of
a complex mixture, such as contained in whole human DNA.
[0040] Samples or specimens include any microsatellite DNA
sequence, whatever the origin, as long as the sequence is
detectably present in a sample. While routine diagnostic tests may
not be able to identify cancer cells in these samples, the
non-invasive method of the present invention identifies neoplastic
cells derived from the primary tumor. The sample of microsatellite
DNA of the subject may be serum, plasma, lymphocytes, urine,
sputum, bile, stool, cervical tissue, saliva, tears, cerebral
spinal fluid, regional lymph node, histopathologic margins, and any
bodily fluid that drains a body cavity or organ. Therefore, the
method provides for the non-invasive detection of various tumor
types including head and neck cancer, lung cancer, esophageal
cancer, stomach cancer, small bowel cancer, colon cancer, bladder
cancer, kidney cancers, cervical cancer and any other organ type
that has a draining fluid accessible to analysis. For example,
neoplasia of regional lymph nodes associated with a primary mammary
tumor can be detected using the method of the invention. Regional
lymph nodes for head and neck carcinomas include cervical lymph
nodes, prelaryngeal lymph nodes, pulmonary juxtaesophageal lymph
nodes and submandibular lymph nodes. Regional lymph nodes for
mammary tissue carcinomas include the axillary and intercostal
nodes. Samples also include urine DNA for bladder cancer or plasma
or saliva DNA for head and neck cancer patients.
[0041] The method of the invention can also be used to detect a
microsatellite DNA sequences associated with a primary tumor by
assaying the surrounding tumor margin. A "tumor margin" as used
herein refers to the tissue surrounding a discernible tumor. In the
case of surgical removal of a solid tumor, the tumor margin is the
tissue cut away with the discernible tumor that appears normal to
the naked eye.
[0042] An allelic imbalance may be detected as a decrease in the
level of DNA corresponding to an allele. The term "decrease in the
level of DNA" refers to the observed difference of the ratio
between the two alleles for a genetic locus. A sample of cells can
have a ratio approaching 1:1 for a subject that lacks the cell
proliferative disorder. The actual ratio for a subject can readily
be determined by analyzing the DNA from the normal (non-tumor)
cells of the tested subject. The level of DNA corresponding to the
allele may be less than 50% of the level of DNA of a corresponding
allele in a microsatellite DNA sample of a subject that lacks the
cell proliferative disorder.
[0043] An allelic imbalance may also be detected as an increase in
the level of DNA corresponding to an allele. The term "increase in
the level of DNA" refers to the observed difference of the ratio
between the two alleles for a genetic locus. A sample of cells will
have a ratio that approaches 1:1 for a subject that lacks the cell
proliferative disorder. Analyzing the DNA from the normal
(non-tumor) cells of a test subject can readily determine the
actual ratio for the subject.
[0044] An increase in the level of DNA may be detected as the
appearance of a new allele. The term "presence of a new allele"
refers both refers to the observed difference of the ratio between
the two alleles for a genetic locus and to genetic instability, the
genetic recombinations that are characteristic of tumor cells and
that result in nucleic acid mutations as described infra. When
there has been an increase in the number of chromosomes for a
member of the chromosomal set, one of the chromosomes may undergo
genetic recombination, so that there will be an addition or
deletion of DNA repeats in the microsatellite DNA sequence. The
mutated microsatellite DNA sequence is therefore a new allele for
that genetic locus, as compared with the normal (non-tumor) cells
of the subject. For example, a tumor cell may have three or more
different alleles for a genetic locus instead of the two alleles
found in the normal (non-tumor) cells. For another example, the
presence of a new allele may correspond to the loss of an allele
found in normal (non-tumor) cells.
[0045] Detection of an allelic imbalance may be performed by
standard methods such as size fractionating the DNA. The term "size
fractionating the DNA" refers to the separation of individual DNA
molecules according to the size of the molecule. Methods of
fractionating the DNA are well known to those of skill in the art.
Fractionating the DNA on the basis of size may be accomplished by
gel electrophoresis, including polyacrylamide gel electrophoresis
(PAGE). For example, the gel may be a denaturing 7 M or 8 M
urea-polyacrylamide-formamide gel. Size fractionating the DNA may
also be accomplished by chromatographic methods known to those of
skill in the art.
[0046] The reaction products containing microsatellite DNA may
optionally be radioactively labeled. Any radioactive label may be
employed which provides an adequate signal. Other labels include
ligands, which can serve as a specific binding pair member for a
labeled ligand, and the like. The labeled preparations are used to
probe nucleic acid by the Southern hybridization technique, for
example. Test nucleotide fragments are transferred to filters that
bind nucleic acid. After exposure to the labeled microsatellite DNA
probe, which will hybridize to nucleotide fragments containing
target nucleic acid sequences, the binding of the radioactive probe
to target nucleic acid fragments is identified by autoradiography
(see Genetic Engineering, 1, ed. Robert Williamson, Academic Press
(1981), pp. 72-81). The particular hybridization technique is not
essential to the invention. Several hybridization techniques are
well known or easily ascertained by one of ordinary skill in the
art. As improvements are made in hybridization techniques, they can
readily be applied in the method of the invention. This technique
provides a further method of identification that can be additional
or an alternative to size fractionation.
[0047] The microsatellite DNA may be amplified before detecting.
The term "amplified" refers to the process of making multiple
copies of DNA from a single molecule of DNA by genetic duplication.
The amplification of DNA may occur in vivo by cellular mechanisms.
The amplification of DNA may also occur in vitro by biochemical
processes known to those of skill in the art. The amplification
agent may be any compound or system that will function to
accomplish the synthesis of primer extension products, including
enzymes. Suitable enzymes for this purpose include, for example, E.
coli DNA polymerase I, Taq polymerase, Klenow fragment of E. coli
DNA polymerase I, T4 DNA polymerase, other available DNA
polymerases, polymerase muteins, reverse transcriptase, ligase, and
other enzymes, including heat-stable enzymes (i.e., those enzymes
that perform primer extension after being subjected to temperatures
sufficiently elevated to cause denaturation). Suitable enzymes will
facilitate combination of the nucleotides in the proper manner to
form the primer extension products that are complementary to each
mutant nucleotide strand. Generally, the synthesis will be
initiated at the 3' end of each primer and proceed in the 5'
direction along the template strand, until synthesis terminates,
producing molecules of different lengths. There may be
amplification agents, however, that initiate synthesis at the 5'
end and proceed in the other direction, using the same process as
described above. In any event, the method of the invention is not
to be limited to the embodiments of amplification described
herein.
[0048] One method of in vitro amplification which can be used
according to this invention is the polymerase chain reaction (PCR)
described in U.S. Pat. Nos. 4,683,202 and 4,683,195. The term
"polymerase chain reaction" refers to a method for amplifying a DNA
base sequence using a heat-stable DNA polymerase and two
oligonucleotide primers, one complementary to the (+)-strand at one
end of the sequence to be amplified and the other complementary to
the (-)-strand at the other end. Because the newly synthesized DNA
strands can subsequently serve as additional templates for the same
primer sequences, successive rounds of primer annealing, strand
elongation, and dissociation produce rapid and highly specific
amplification of the desired sequence. The polymerase chain
reaction is used to detect the existence of the defined sequence in
the microsatellite DNA sample. Many polymerase chain methods are
known to those of skill in the art and may be used in the method of
the invention. For example, DNA can be subjected to 30 to 35 cycles
of amplification in a thermocycler as follows: 95.degree. C. for 30
sec, 52.degree. to 60.degree. C. for 1 min, and 72.degree. C. for 1
min, with a final extension step of 72.degree. C. for 5 min. For
another example, DNA can be subjected to 35 polymerase chain
reaction cycles in a thermocycler at a denaturing temperature of
95.degree. C. for 30 sec, followed by varying annealing
temperatures ranging from 54-58.degree. C. for 1 min, an extension
step at 70.degree. C. for 1 min and a final extension step at
70.degree. C.
[0049] Exemplary target nucleotide sequences of the invention, to
which complementary oligonucleotide primers hybridize, include the
following:
1 5'-CTTGTGTCCCGGCGTCTG-3' SEQ ID NO: 1 5'-CAGCCCAGCAGGACCAGTA-3'
SEQ ID NO: 2 5'-TGGTAACAGTGGAATACTGAC-3' SEQ ID NO: 3
5'-ACTGATGCAAAAATCCTCAAC-3' SEQ ID NO: 4
5'-GATGGGCAAACTGCAGGCCTGGGAAG-3' SEQ ID NO: 5
5'-GCTACAAGGACCCTTCGAGCCCCGTTC-3' SEQ ID NO: 6
5'-GATGGTGATGTGTTGAGACTGGTG-3' SEQ ID NO: 7
5'-GAGCATTTCCCCACCCACTGGAGG-3' SEQ ID NO: 8
5'-GTTCTGGATCACTTCGCGGA-3' SEQ ID NO: 9 5'-TGAGGATGGTTCTCCCCAAG-3'
SEQ ID NO: 10 5'-AGTGGTGAATTAGGGGTGTT-3' SEQ ID NO: 11
5'-CTGCCATCTTGTGGAATCAT-3' SEQ ID NO: 12
5'-CTGTGAGTTCAAAACCTATGG-3' SEQ ID NO: 13
5'-GTGTCAGAGGATCTGAGAAG-3' SEQ ID NO: 14
5'-GCACGCTCTGGAACAGATTCTGGA-3' SEQ ID NO: 15
5'-ATGAGGAACAGCAACCTTCACAGC-3' SEQ ID NO: 16
5'-TCACTCTTGTCGCCCAGATT-3' SEQ ID NO: 17 5'-TATAGCGGTAGGGGAGATGT-3'
SEQ ID NO: 18 5'-TGCAAGGAGAAAGAGAGACTGA-3' SEQ ID NO: 19
5'-AACAGGACCACAGGCTCCTA-3' SEQ ID NO: 20
5'-TCTCTTTCTTTCCTTGACAGGGTC-3' SEQ ID NO: 21
5'-CAGTGTGGTCCCAAATTTGAAATGG-3' SEQ ID NO: 22
5'-GTGCTGACTAGGGCAGCTT-3' SEQ ID NO: 23 5'-TGTGACCTGCACTCGGAAGC-3'
SEQ ID NO: 24 5'-CCTTTCCTTCCTTCCTTCC-3' SEQ ID NO: 25
5'-CACAGTCAGGTCAGGCTATCAG-3' SEQ ID NO: 26
5'-TTTTTGAGATAGAGTCTCACTGTG-3' SEQ ID NO: 27
5'-CCACAGTCTAAGCCAGTCTGA-3' SEQ ID NO: 28
5'-GAATTTTGCTCTTGTTGCCCAG-3' SEQ ID NO: 29
5'-AGACTGAAGTCAATGAACAACAAC-3' SEQ ID NO: 30
5'-GGCTGTGAACATGGCCTAGGTC-3' SEQ ID NO: 31
5'-TTGGGGTGGTGCCAATGGATGTC-3' SEQ ID NO: 32
[0050] Exemplary oligonucleotide primers, of the invention, include
the following:
2 5'-CAGACGCCGGGACACAAG-3' SEQ ID NO: 33 5'-TACTGGTCCTGCTGGGCTG-3'
SEQ ID NO: 34 D21S1245(F) 5'-GTCAGTATTACCCTGTTACCA-3' SEQ ID NO: 35
D21S1245(R) 5'-GTTGAGGATTTTTGCATCAGT-3' SEQ ID NO: 36
5'-CTTCCCAGGCCTGCAGTTTGCCCATC-3' SEQ ID NO: 37
5'-GAACGGGGCTCGAAGGGTCCTTGTAGC-3' SEQ ID NO: 38 DRPLA(F)
5'-CACCAGTCTCAACACATCACCATC-3' SEQ ID NO: 39 DRPLA(R)
5'-CCTCCAGTGGGTGGGGAAATGCTC-3' SEQ ID NO: 40
5'-TCCGCGAAGTGATCCAGAAC-3' SEQ ID NO: 41 5'-CTTGGGGAGAACCATCCTCA-3'
SEQ ID NO: 42 D14S50(F) 5'-AACACCCCTAATTCACCACT-3' SEQ ID NO: 43
D14S50(R) 5'-ATGATTCCACAAGATGGCAG-3' SEQ ID NO: 44 FgA(F)
5'-CCATAGGTTTTGAACTCACAG-3' SEQ ID NO: 45 FgA(R)
5'-CTTCTCAGATCCTCTGACAC-3' SEQ ID NO: 46 D20548(F)
5'-TCCAGAATCTGTTCCAGAGCGTGC-3' SEQ ID NO: 47 D20548(R)
5'-GCTGTGAAGGTTGCTGTTCCTCAT-3' SEQ ID NO: 48
5'-AATCTGGGCGACAAGAGTGA-3' SEQ ID NO: 49 5'-ACATCTCCCCTACCGCTATA-3'
SEQ ID NO: 50 5'-TCAGTCTCTCTTTCTCCTTGCA-3' SEQ ID NO: 51
5'-TAGGAGCCTGTGGTCCTGTT-3' SEQ ID NO: 52 D8S3G7(F)
5'-GACCCTGTCAAGGAAAGAAAGAGA-3' SEQ ID NO: 53 D8S3G7(R)
5'-CCATTTCAAATTTGGGACCACACTG-3' SEQ ID NO: 54 THO(F)
5'-AAGCTGCCCTAGTCAGCAC-3' SEQ ID NO: 55 THO(R)
5'-GCTTCCGAGTGCAGGTCACA-3' SEQ ID NO: 56 D115488(F)
5'-mGGAAGGAAGGAAGGAAAGG-3' SEQ ID NO: 57 D115488(R)
5'-CTGATAGCCTGACCTGACTGTG-3' SEQ ID NO: 58 D135802(F)
5'-CACAGTGAGACTCTATCTCAAAAA-3' SEQ ID NO: 59 D135802(R)
5'-TCAGACTGGCTTAGACTGTGG-3' SEQ ID NO: 60 D175695(F)
5'-CTGGGCAACAAGAGCAAAATTC-3' SEQ ID NO: 61 D175695(R)
5'-mGTTGTTGTTCATTGACTTCAGTCT-3' SEQ ID NO: 62 D175654(F)
5'-GACCTAGGCCATGTTCACAGCC-3' SEQ ID NO: 63 D175654(R)
5'-GACATCCATTGGCACCACCCCAA-3' SEQ ID NO: 64
[0051] Those of ordinary skill in the art will know of various
amplification methodologies which can also be utilized to increase
the copy number of target nucleic acid. Microsatellite DNA sequence
detected in the method of the invention can be further evaluated,
detected, cloned, sequenced, and the like, either in solution or
after binding to a solid support, by any method usually applied to
the detection of a specific DNA sequence such as another polymerase
chain reaction, oligomer restriction (Saiki et al., Bio/Technology
3: 1008-1012 (1985)), allele-specific oligonucleotide (ASO) probe
analysis (Conner et al., Proc. Natl. Acad. Sci. USA 80: 278 (1983),
oligonucleotide ligation assays (OLAs) (Landegren et al., Science
241: 1077 (1988)), and the like. Molecular techniques for DNA
analysis have been reviewed (Landegren et al, Science, 242: 229-237
(1988)). Following DNA amplification, the reaction product may be
detected by Southern blot analysis, without using radioactive
probes. In such a process, for example, a small sample of DNA
containing a very low level of microsatellite DNA nucleotide
sequence is amplified, and analyzed via a Southern blotting
technique. The use of non-radioactive probes or labels is
facilitated by the high level of the amplified signal. In a
preferred embodiment of the invention, one nucleoside triphosphate
is radioactively labeled, thereby allowing direct visualization of
the amplification product by autoradiography. In another
embodiment, amplification primers are fluorescent labeled and run
through an electrophoresis system. Visualization of amplified
products is by laser detection followed by computer assisted
graphic display.
[0052] In another embodiment, the invention is a method for
detecting a cell proliferative disorder in a subject. The method
detects genetic instability in a sample of microsatellite DNA of
the mammal as an indication of a cell proliferative disorder. As
used herein, the term "genetic instability" refers to genetic
recombinations which are characteristic of tumor cells and which
result in nucleic acid mutations. Such mutations include the
deletion and addition of nucleotides. The genetically unstable
sequences of the invention are preferably microsatellite DNA
sequences which, by definition, are small tandem repeat DNA
sequences.
[0053] Genetic recombination tends to occur most frequently at
regions of the chromosome where the DNA is homologous (where the
DNA has a high degree of sequence similarity). Where a DNA sequence
is repetitive, the DNA homology is greater. The DNA homology occurs
not only at the same genetic locus on the other pair of
chromosomes, but also on other genetic loci or within the same
locus on the same chromosome. In normal (non-tumor cells) this
genetic recombination tends to be suppressed. Tumor cells, however,
characteristically undergo increased genetic recombination. Where a
DNA sequence is repetitive, genetic recombination can result in the
loss of repeat DNA sequences or the gain of repeat DNA sequences at
a genetic locus.
[0054] When the microsatellite DNA repeat is larger, it is more
likely that the microsatellite DNA locus will have mutations. A
trinucleotide repeat is more likely to have deletions or additions
than a dinucleotide repeat. A regular repeat, such as AATAATAAT is
more likely to have mutations than a sequence which contains
interruptions in the repeat sequence, e.g., AATGACAATAAT.
Consequently, those of ordinary skill in the art can readily
identify other target nucleic acid sequences by considering the
size of the candidate sequence and whether the sequence is
uninterrupted without resorting to undue experimentation. Other
microsatellite DNA markers will be known by the criteria described
herein and are accessible to those of skill in the art. Smaller
microsatellite DNA markers including dinucleotide and
mononucleotide repeats will also be useful for this analysis.
[0055] The genetic instability may be detected as an amplification
of nucleotide repeats in the DNA. The term "amplification of
nucleotide repeats" refers to a mutation in the sequence of the
microsatellite DNA wherein the resulting microsatellite DNA
sequence has more DNA repeats than the sequence found in normal
(non-tumor) cells. Where the normal cell microsatellite DNA has a
sequence (X).sub.n1, X is the number of nucleotides, and n1 and n2
are numbers of repeats, the resulting microsatellite DNA sequence
is (X).sub.n1+n2.
[0056] The instability may be detected as a deletion of nucleotide
repeats in the DNA. The term "deletion of nucleotide repeats"
refers to a mutation in the sequence of the microsatellite DNA
wherein the resulting microsatellite DNA sequence has fewer DNA
repeats than the sequence found in normal (non-tumor) cells. Where
the normal cell microsatellite DNA has a sequence (X).sub.n1, X is
the number of nucleotides, n1 and n2 are numbers of repeats, and n1
is greater than n2, the resulting microsatellite DNA sequence is
(X).sub.n1-n2.
[0057] The instability may be detected when the DNA is amplified
before detecting. The amplification may be accomplished by the
polymerase chain reaction, as described supra. Those of skill in
the art will know of other amplification methods which can increase
the copy number of target nucleic acid.
[0058] The genetic instability may be detected when the cell
proliferative disorder is not due to a repair gene defect. The term
"repair gene defect" refers to a defect in a gene coding for any of
a number of processes to repair damaged DNA. In gene repair, the
damaged portions of the DNA molecule are removed by enzymes (each
enzyme coded for by a repair gene), leaving holes where bases
should be. Then other enzymes (also coded for by repair genes)
remove an entire segment of DNA, in the middle of which was the
hole or holes. A DNA polymerase (coded for by repair genes) then
fills the gap with nucleotide bases, based on what bases are on the
opposite strand of DNA. Finally a ligase enzyme (coded for by
repair genes) seals the phosphate backbone back together.
[0059] Another type of DNA repair is error-prone repair (SOS
repair), which occurs when both nucleotides in a base pair are
missing, such that it is not possible to maintain accuracy. The
enzymes and other proteins which mediate error-prone repair are
coded for by repair genes. Still another type of DNA repair is
excision repair, where the damaged portion of DNA is excised, or
removed, then the removed part is recopied from the other undamaged
strand by DNA polymerase enzymes, and finally the replacement part
is attached to the site by DNA ligase enzymes. The enzymes and
other proteins which mediate excision repair are also coded for by
repair genes.
[0060] Those of skill in the art will be familiar with those genes
which code for the processes which repair damaged DNA. Those of
skill in the art will also be able to identify these genes by their
chromosomal location and by the methods of DNA amplification and
size fractionation.
[0061] The genetic instability may be detected when the cell
proliferative disorder is a neoplasm. Neoplasms are described
supra.
[0062] The microsatellite DNA may be from a locus that has been
used in genetic mapping. Among the loci may be one or more of the
following: DRPLA, UT762, IFNA, D9S200, D9S156, D3S1284, D3S1238,
CHRNB1, D17S86, D9S747, D9S171, D16S476, D4S243, D14S50, D21S1245,
FgA, D8S3G7, THO, D115488, D135802, D175695, D175654, and
D20548.
[0063] The term "oligonucleotide primer" refers to a sequence of
two or more deoxyribonucleotides or ribonucleotides, preferably at
least eight, which sequence is capable of initiating synthesis of a
primer extension product that is substantially complementary to a
target nucleic acid strand. The oligonucleotide primer typically
contains fifteen to twenty-two or more nucleotides, although it may
contain fewer nucleotides if the primer is complementary, so as to
specifically allow the amplification of the specifically desired
target nucleotide sequence.
[0064] The oligonucleotide primers for use in the invention may be
prepared using any suitable method, such as conventional
phosphotriester and phosphodiester methods or automated embodiments
thereof. In one such automated embodiment, diethylphosphoramidites
are used as starting materials and may be synthesized as described
by Beaucage et al., Tetrahedron Letter, 22: 1859-1862 (1981). One
method for synthesizing oligonucleotides on a modified solid
support is described in U.S. Pat. No. 4,458,066. The exact length
of primer will depend on many factors, including temperature,
buffer, and nucleotide composition. The primer must prime the
synthesis of extension products in the presence of the inducing
agent for amplification.
[0065] Primers used according to the method of the invention are
complementary to each strand of mutant nucleotide sequence to be
amplified. The term "complementary" means that the primers must
hybridize with their respective strands under conditions which
allow the agent for polymerization to function. In other words, the
primers that are complementary to the flanking sequences hybridize
with the flanking sequences and permit amplification of the
nucleotide sequence. Preferably, the 3' terminus of the primer that
is extended has perfectly base paired complementarity with the
complementary flanking strand.
[0066] The term "flanks nucleotide repeats" refers to those DNA
sequences on chromosome that are upstream (5') or downstream (3')
to the DNA sequence to be amplified. The nucleotide repeat sequence
to be amplified is preferably a microsatellite DNA sequence. For
example, when the nucleotide repeat sequence to be amplified is
double stranded, a first sequence that is 5' to the nucleotide
repeat sequence and a second sequence that is 5' to the nucleotide
repeat sequence on the complementary strand flank the
microsatellite DNA sequence.
[0067] The nucleotide sequences that flank nucleotide repeats,
i.e., the nucleotide sequences to which the oligonucleotide primers
hybridize, may be selected from among the following nucleotide
sequences: SEQ ID NO:1-32.
[0068] When it is desirable to amplify the target nucleotide
sequence, such as a microsatellite DNA sequence, before detection,
oligonucleotides can be used as the primers for amplification. The
oligonucleotide primers are designed based upon identification of
the nucleic acid sequence of the flanking regions contiguous with
the microsatellite DNA. One skilled in the art will be able to
generate primers suitable for amplifying target sequences of
additional nucleic acids, such as those flanking loci of known
microsatellite DNA sequences, using routine skills known in the art
and the teachings of this invention.
[0069] The oligonucleotide primers used in the amplification may be
selected from among the following primers: SEQ ID NO:33-64.
[0070] In another embodiment, the invention provides a kit for
detecting a mammalian cell proliferative disorder. The kit
comprises an oligonucleotide primer that is complementary to a
nucleic acid sequence that flanks nucleotide repeats of
microsatellite DNA. Such a kit may also include a carrier means
being compartmentalized to receive in close confinement one or more
containers such as vials, tubes, and the like, each of the
containers comprising one of the separate elements to be used in
the method. For example, one of the containers may include
amplification primers for a microsatellite DNA locus or a
hybridization probe, all of which can be detectably labeled. If
present, a second container may comprise a lysis buffer.
[0071] The kit may also have containers containing nucleotides for
amplification of the target nucleic acid sequence which may or may
not be labeled, or a container comprising a reporter, such as a
biotin-binding protein, such as avidin or streptavidin, bound to a
reporter molecule, such as an enzymatic, florescent, or
radionuclide label. The term "detectably labeled
deoxyribonucleotide" refers to a means for identifying
deoxyribonucleotide. The detectable label may be a radiolabeled
nucleotide. The detectable label may be a small molecule covalently
bound to the nucleotide where the small molecule is recognized by a
well-characterized large molecule. Examples of these small
molecules are biotin, which is bound by avidin, and thyroxin, which
is bound by anti-thyroxin antibody. Other methods of labeling are
known to those of ordinary skill in the art, including fluorescent
compounds, chemiluminescent compounds, phosphorescent compounds,
and bioluminescent compounds.
[0072] In general, the primers used according to the method of the
invention embrace oligonucleotides of sufficient length and
appropriate sequence which provide specific initiation of
polymerization of a significant number of nucleic acid molecules
containing the target nucleic acid under the conditions of
stringency for the reaction utilizing the primers. In this manner,
it is possible to selectively amplify the specific target nucleic
acid sequence containing the nucleic acid of interest.
Oligonucleotide primers used according to the invention are
employed in any amplification process that produces increased
quantities of target nucleic acid.
[0073] The above disclosure generally describes the present
invention. A more complete understanding can be obtained by
reference to the following specific examples. These examples are
provided herein for purposes of illustration only and are not
intended to limit the scope of the invention.
EXAMPLE 1
Molecular Detection of Primary Bladder Cancer by Microsatellite DNA
Analysis
[0074] The purpose of this Example is to show that microsatellite
DNA markers are useful as clonal markers for the detection of human
cancer, because simple DNA repeat mutations can be readily detected
in clinical samples by the polymerase chain reaction. In this
Example, the feasibility of polymerase chain reaction-based
microsatellite DNA analysis of DNA from urine sediment is shown by
correctly identifying nineteen of twenty patients with primary
bladder tumors by this approach. In contrast, using urine cytology,
only nine of eighteen affected patients were detected.
[0075] Sixty trinucleotide and tetranucleotide markers in the DNA
from fifty anonymous primary bladder cancers were screened. The
screening was done in the following manner: Frozen tumor tissue was
cut into 10 .mu.m sections. All samples, including lymphocytes,
were digested with 1% SDS-proteinase K at 60.degree. C. for 5 hr.
DNA was extracted by ethanol precipitation. Urine samples were spun
at 3000 g for 5 min and washed twice with phosphate-buffered
saline. Each polymerase chain reaction mixture (25 .mu.l) contained
50 ng of DNA template. Primers were obtained from Research Genetics
(Huntsville, Ala.) or synthesized from sequences in the Genome
Database. For microsatellite DNA analysis, one primer was labeled
with T4 polynucleotide kinase (New England Biolabs) and
[.gamma.-.sup.32P]-adenosine triphosphate (New England Nuclear).
DNA was subjected to 30 to 35 cycles of amplification in a Hybaid
(Middlesex, UK) Omnigene TR3 SM2 Thermocycler as follows:
95.degree. C. for 30 sec, 52.degree. to 60.degree. C. for 1 min,
and 72.degree. C. for 1 min, with a final extension step of
72.degree. C. for 5 min. Polymerase chain reaction products were
separated by electrophoresis in denaturing 8 M
urea-polyacrylamide-formamide gels, which were then subjected to
autoradiography.
[0076] Of the screened primary bladder cancers, 40 (80%) contained
at least one marker alteration when compared with the DNA from
matched normal lymphocytes. Calculations showed that a panel of the
ten most useful markers would theoretically detect mutations in 52%
of all cancers. The calculation was done as follows: The total
mutations for each marker tested in all tumors were tabulated and
then grouped, in descending order, from the markers most
susceptible in mutations that would empirically detect the greater
number of primary tumors. Thus, the ten most susceptible markers
empirically identified at least on alteration in twenty-six of
fifty tumors (52%), eleven identified 56% of tumors, twelve
identified 60%, thirteen identified 62%, and so forth. Some markers
identified mutations only in tumors previously identified by
another marker, and 20% of tumors did not demonstrate a single
alteration with any marker tested.
[0077] Twenty-five patients who were screened for primary bladder
cancers presented with symptoms suggestive of bladder cancer (for
example, gross hematuria) and were found to harbor suspicious
lesions at cystoscopy. Urine samples were collected (before
cystoscopy) and were then distributed in blinded fashion for
microsatellite DNA analysis and routine urine cytology. Then, the
DNA in the urine sediment from these twenty-five patients with
suspicious bladder lesions and from five controls (patients without
evidence of bladder cancer) was tested. Urine and lymphocyte DNA
from each patient were amplified by polymerase chain reaction, and
polymorphic alleles were compared at the ten preselected
microsatellite DNA loci. The urine DNA of ten patients contained a
microsatellite DNA mutations (expansion or deletion of a repeat
unit), in close agreement with the frequency expected on the basis
of the calculations.
[0078] In addition to microsatellite DNA mutations, primary tumors
often harbor chromosomal deletions at suppressor gene loci that are
manifested as loss of heterozygosity loss of heterozygosity and are
readily detected by microsatellite DNA analysis. Notably, eighteen
urine DNA samples also demonstrated loss of heterozygosity,
particularly with marker D9S747 from chromosome 9p21. This result
is consistent with the observation that loss of chromosome 9 occurs
frequently in bladder cancer. Analysis of three additional
dinucleotide markers on chromosome 9p21 (D9S171, D9S162, and IFNA)
for loss of heterozygosity confirmed the presence of deletions in
urine samples that demonstrated loss of chromosome 9 with marker
D9S747.
[0079] That the genetically altered alleles were derived from
exfoliated cancer cells was confirmed by two methods:
[0080] First, the primary tumors from biopsies of fifteen of the
twenty cancer patients (in five cases, there was insufficient
biopsy material for this analysis) were examined. In all patients,
the same microsatellite DNA mutations and loss of heterozygosity
patterns detected in the urine were also detected in the primary
tumor. However, in two patients, the urine samples showed loss of
heterozygosity or microsatellite DNA mutations that were not
present in the biopsies. In both cases, loss of heterozygosity in
at least one locus (and loss of the identical allele) was shared
between the urine sediment and the primary tumor. Conceivably, the
urine sample may have contained a more advanced tumor cell clone
that was derived from the same progenitor cell but was not sampled
by the small biopsy of the tumor.
[0081] Second, cells from the same urine samples were then examined
by light microscopy. Cytologic analysis was performed in a blinded
fashion, following normal clinical procedures in samples from
eighteen of the twenty patients with bladder cancer and from three
of the five patients with suspicious lesions but without neoplasia.
Those normal clinical procedures are as follows: Approximately 50
cm.sup.3 of urine was obtained and concentrated by centrifugation
on cytospin glass slides or Millipore filters (Burlington, Mass.).
Cells were stained by Papanicolaou stain and visualized under
microscopy. Standard morphologic criteria were used to establish
the presence of neoplastic cells. Neoplastic cells were identified
by cytology in nine of the eighteen patients for whom molecular
analysis was positive and in one patient for whom molecular
analysis was negative.
[0082] Twenty of the twenty-five patients had histologically
confirmed bladder cancer. Overall, microsatellite DNA analysis with
the thirteen markers detected genetic mutations in nineteen of
these twenty cancer patients. Of four patients with inflammation
that prompted cystoscopy, two showed molecular changes (loss of
heterozygosity, genetic instability, or both) in the urine, and
both had bladder lesions containing atypical cells that were
suspicious but not diagnostic for cancer. None of the five patients
without neoplasia (controls) showed any microsatellite DNA
mutations (see FIGS. 1 and 2).
[0083] This Example demonstrates that microsatellite DNA analysis
can be a powerful tool in the detection of primary bladder cancer.
The ease of loss of heterozygosity detection in urine sediment is
consistent with analysis on urine samples by fluorescence in situ
hybridization (FISH). Moreover, molecular analysis of patients with
multiple tumors has demonstrated that these multiple tumors
appeared to arise from a single progenitor cell that seeded and
populated the bladder mucosa, potentially accounting for the high
risk of recurrence in these patients. These observations are
compatible with the hypothesis that large areas of transformed
bladder mucosa can exist in patients with small neoplasms. Other
factors may also contribute to the enrichment of tumor cells in
urine; for example, more tumor cells than normal cells may survive
storage. In addition, as tumor surfaces are composed of actively
growing cell populations that clonally expand through mechanisms
such as loss of adhesion, it is possible that these cells are more
readily shed into the urine.
[0084] These microsatellite DNA markers enabled the detection of
95% of the bladder cancers in this study, but as new markers are
identified, the approach can be expanded and improved. Despite an
expected identification of only .about.50% of cases, the
identification of loss of heterozygosity in addition to
microsatellite DNA mutations greatly improved the detection
strategy. An adenocarcinoma of the prostatic fossa was also
identified, indicating that markers commonly deleted in other
genitourinary tract neoplasms may facilitate the detection of other
neoplasms that exfoliate cells into urine sediment.
[0085] Finally, our approach highlights the immediate utility of
studies that demonstrate loss of heterozygosity in human cancer and
of the development of molecular progression models for clinical
detection. In most of the cases in this study, morphologic and
cytologic analyses were not diagnostic. Molecular analysis reliably
detected tumors of all grades and stages, including those often
missed by cytology. In principle, this molecular approach can be
performed at approximately one-third the cost of cytology and does
not require exhaustive expert interpretation.
[0086] Moreover, the entire assay is amenable to nonradioactive,
non-gel separation techniques and potentially could lead to a
reliable, yet inexpensive, molecular screening test.
EXAMPLE 2
Microsatellite DNA Alterations in Serum DNA of Head and Neck Cancer
Patients
[0087] The purpose of this Example is to show that microsatellite
DNA analysis of serum represents a novel method for the detection
of circulating tumor cell DNA. Lymphocyte, serum and tumor DNA were
retrospectively analyzed from twenty-one head and neck cancer
patients. Head and neck cancer remains a morbid and often fatal
disease. Large tumor bulk and tumor extension are predictors of
local regional recurrence and poor outcome. Molecular detection of
occult neoplastic cells in surrounding surgical margins is a strong
predictor of local regional recurrence resulting in a significant
decrease in overall survival. In this Example, twenty-one patients
were followed from initial diagnosis of transitional cell carcinoma
with microsatellite DNA analysis of urine DNA at the time of
cystoscopic evaluations scheduled at routine intervals. In almost
all cases, DNA-based analysis correlated precisely with clinical
findings at cystoscopy and subsequent histopathology. In two cases,
DNA analysis correctly predicted the presence of a tumor several
months before the lesion was detected during examination with a
cystoscope.
[0088] These twenty-one patients were chosen from our tumor bank
because all three DNA sources were available for complete analysis,
and the serum samples had been collected before surgical resection
of head and neck cancer. Twelve microsatellite DNA markers were
selected to detect shifts of loss of heterozygosity. Eight markers
were chosen on 9p, 3p and 17p, because these chromosomal arms show
the highest percentage of loss of heterozygosity and appear to
harbor tumor suppressor gene loci involved early in the progression
of head and neck cancer. Furthermore, two trinucleotide (D1S50 and
DRPLA) and two tetranucleotide (D21S1245 and FgA) markers,
recognized as being prone to microsatellite DNA instability and
located on loci commonly altered in cancers, were used in the study
for increased sensitivity in the detection of shifts. Loss of
heterozygosity was scored if the allele signal was reduced to less
then 50% of control intensity. Shifts were called if there was an
obvious new allele compared with normal (non-tumor) lymphocyte
DNA.
[0089] Sample collection and DNA isolation was accomplished as
follows: Tumors obtained fresh from surgical resection and blood by
venipuncture from head and neck cancer patients were collected from
patients at the Johns Hopkins University Medical Institutions with
prior consent. To obtain, serum, clotted blood specimens were
centrifuged at low speed for 5 min, and the serum was stored at
-80.degree. C. before DNA extraction. Tumor tissue was frozen and
microdissected. Lymphocytes, tumor tissue and serum were digested
in SDS and proteinase K at 48.degree. C. overnight, followed by
phenol/chloroform extraction and ethanol precipitation of DNA. The
mean concentration of DNA in the cancer patients was 110.+-.50 ng
per ml serum, and 10 .mu.l was usually sufficient for robust
microsatellite DNA analysis. The concentration of serum DNA from
normal controls ranges from 0 to 100 ng/ml.
[0090] Polymerase chain reaction amplification was performed as
follows: Oligonucleotide markers for microsatellite DNA analysis
were obtained from Research Genetics (Huntsville, Ala.) and
included IFNA, D9S156, D9S161, D9S200 on 9p; D3S1238, D3S1284 on
3p; D17S786 and CHRNB1 on 17p. Trinucleotide and tetranucleotide
primers used included the following:
3 for D14S50: D14S50(F) 5'-AACACCCCTAATTCACCACT-3' (SEQ ID NO: 43)
D14S50(R) 5'-ATGATTCCACAAGATGGCAG-- 3' (SEQ ID NO: 44) for
D21S1245: D21S1245(F) 5'-GTCAGTATTACCCTGTTACCA-3' (SEQ ID NO: 35)
D21S1245(R) 5'-GTTGAGGATTTTTGCATCAGT-3' (SEQ ID NO: 36) for DRPLA:
DRPLA(F) 5'-CACAGTCTCAACACATC-3' (SEQ ID NO: 39) DRPLA(R)
5'-CCTCCAGTGGGTGGGGAAATGCCTC-3' (SEQ ID NO: 40) for FgA: FgA(F)
5'-CCATAGGTTTTGAACTCACA- G-3' (SEQ ID NO: 45) FgA(R)
5'-CTTCTCAGATTCCTTCTTGA- CAC-3' (SEQ ID NO: 46)
[0091] One primer from each set was end labeled with
(.gamma.-.sup.32P) ATP (Amersham) using T4 polynucleotide kinase
(New England Biolabs). Polymerase chain reaction amplification was
performed with 30-60 ng DNA was described previously. Products were
separated in 8% denaturing urea-polyacrylamide-formamide gels
followed by autoradiography. Loss of heterozygosity was called if
the ratio of one allele was significantly decreased (>50%) in
tumor or serum DNA compared with normal (non-tumor) lymphocyte
DNA.
[0092] A tumor-specific microsatellite DNA shift, represented by a
novel allele after gel electrophoresis, can still be seen when
tumor DNA is diluted between 1:500 to 1:1000 with normal DNA.
Shifts derived from primary tumor cell DNA in the serum might have
been expected. However, there was a surprisingly clear loss of
heterozygosity in serum DNA. The first patient that had such
provocative results was an 80-year-old man diagnosed with
T.sub.3N.sub.cM.sub.0 glottic cancer who underwent a total
laryngectomy in January 1993. He had no evidence of disease on
three months' follow-up. However, in September 1993, he was
diagnosed with recurrence of tumor in the right neck with the mass
surrounding the right carotid artery, and he died of regional
disease in November 1994. The serum DNA of this patient displayed a
clear loss of heterozygosity. This result was unexpected and
reminiscent of the clear loss of heterozygosity and shifts seen in
the urine of patients diagnosed with bladder cancer.
[0093] After microsatellite DNA analysis of all specimens was
completed, clinical data were correlated with the results. Clinical
correlation was performed as follows: Clinical outcome data was
obtained from the Johns Hopkins Head and Neck Cancer Tumor Registry
and by chart review. Fisher's exact test was used to compare
results from plasma analysis with clinical outcome parameters (see
FIGS. 3 and 4).
[0094] All six patients that had microsatellite DNA mutations in
the serum DNA demonstrated identical mutations in the primary tumor
DNA. Four out of six patients displayed mutations in more than one
locus, and all of these patients had advanced disease (stage
III-IV). In this small group, four patients went on to die from
cancer, one patient has terminal cancer with metastases and one
patient has no evidence of disease at three years' follow-up. Five
patients had nodal metastases and three of them later developed
distant metastases, one patient to lung and bone and the other two
patients to lung and liver.
[0095] Conversely, another nine patients had advanced stage cancer,
but no microsatellite DNA mutations in their serum DNA. Six of
these patients had successful resections and were free of disease
on long-term follow-up (more than one year); two of them died
within two years diagnosis from regional recurrence and one was
lost to follow-up. Seven of these patients, including those with a
good prognosis, exhibited loss of heterozygosity or shifts in their
primary tumor DNA but had no evidence of mutations in serum DNA.
Lack of positive findings in serum DNA was also seen in six out of
twenty-one patients that had stage I and II cancer, all with good
prognoses except for one patient who had a recurrence seven years
later and died. Three patients displayed no microsatellite DNA
mutations in their primary tumor DNA with the tested markers. The
data are statistically significant for a positive serum test as
predictor of future distant metastases by the Fisher's exact test
(P=0.015); nevertheless, any conclusions of predictive ability or
clinical utility require verification with larger populations and
well-defined cohorts.
[0096] The results of this Example support the idea of tumor DNA
enrichment in blood serum and plasma. Identification of clear loss
of heterozygosity strongly favors the hypothesis that tumor DNA is
enriched and, in fact, the predominant form of DNA in the plasma.
Such analysis of plasma DNA will be useful in follow-up of cancer
patients receiving medical or surgical treatment. Moreover, serum
microsatellite DNA mutations were always identical to mutations in
the primary tumor DNA. The higher frequency of plasma mutations in
small cell lung cancer may reflect the much higher frequency of
clinical metastases in SCLC patients compared with head and neck
cancer patients. In bulky head and neck tumors, cell lysis by
necrosis or even apoptosis leads to the release of naked DNA into
the circulation. For large tumors, this phenomenon may occur more
frequently because of local angiogenesis and necrosis. Although a
surprising finding, tumor DNA readily survives in various bodily
fluids including urine, stool and sputum.
EXAMPLE 3
Detection of Bladder Cancer Reoccurrence by Microsatellite DNA
Analysis of Urine
[0097] The purpose of this Example was to demonstrate that the
microsatellite DNA analysis method can be used for following-up
patients with transitional cell carcinoma. A reliable, non-invasive
method for monitoring patients with transitional cell carcinoma of
the bladder would be of great clinical benefit. In this Example,
serial urine samples were tested from twenty-one patients who had
been treated for bladder cancer with twenty polymorphic
microsatellite DNA markers in a blinded fashion. Recurrent lesions
were detected in ten out of eleven patients and correctly predicted
the existence of a neoplastic cell population in the urine of two
patients, four and six months before cystoscopic evidence of the
tumor. The assay was negative in ten of ten patients who had no
evident cancer. This Example shows that microsatellite DNA analysis
of urine sediment represents a novel and potentially powerful
clinical tool for the detection of recurrent bladder cancer.
[0098] The microsatellite DNA analysis was done as follows: DNA was
tested from the urine of twenty-one patients with a panel of twenty
microsatellite DNA markers immediately after the initial diagnosis
of a primary bladder tumor. This panel of markers comprised
thirteen markers used in our initial study and seven additional
selected markers. Eleven of these patients were tested in Example 1
and were included here for monitoring of tumor recurrence.
[0099] Initial urine samples (from before resection) were available
from twenty of these twenty-one patients with transitional cell
carcinoma. Tissue and urine specimens were performed as follows:
Twenty patients with histologically confirmed bladder carcinoma and
one patient with transitional cell carcinoma of the renal pelvis
were enrolled into the study. Mean age at time of diagnosis was
68.2 years (50-86), the male/female ratio was 2:1, and the patients
were followed for a mean of eight months (3-18) at our institution.
Venous blood (10 ml) was obtained from every patient for extraction
of normal (germline) DNA to be used as a control. Urine (50 ml) was
collected from each patient before surgical intervention
(transurethral resection or biopsy) and specimens from the initial
surgery or biopsy were frozen at -70.degree. C. immediately before
each follow-up cystoscopy, another 50 ml of urine was obtained from
every patient for analysis. The initial urine sample at first
diagnosis was not available from one patient. All tumors were
diagnosed according to the criteria of American Joint Committee on
Cancer. Urine for cytology was prepared as described previously and
cells were stained with standard Papanicolaou stain. The final
diagnosis from cytopathology (at the Johns Hopkins Hospital) was
entered in the study.
[0100] DNA extraction was performed as follows: Erythrocytes were
lysed by subjecting the blood to TM-solution (5 mM MgCl, 20 mM Tris
buffer), and samples were spun at 3000 r.p.m. for 10 min in order
to obtain a leukocyte pellet. Urine was also spun at 3000 r.p.m.
and the pellet was washed with phosphate-buffered saline. Tumor
specimens were cut into 7-.mu.m sections and standard hematoxylin
and eosin staining was performed. After confirmation of the
diagnosis, neoplastic tissue was microdissected. This material, as
well as the leukocytes and the urine cell pellet, were digested
with 1% SDS and 50 .mu.g/ml proteinase K for 12 hr at 48.degree. C.
DNA was obtained from the samples by phenol-chloroform extraction
and ethanol precipitation.
[0101] Microsatellite DNA analysis was performed as follows: DNA
derived from leukocytes, urine and tumor was analyzed using a panel
of twenty microsatellite DNA markers on different chromosomes
(Research Genetics, Huntsville, Ala., and Oncor, Gaithersburg,
Md.). This panel contained the thirteen markers used in our earlier
study and seven new markers that revealed a high rate of loss of
heterozygosity and shifts in primary bladder tumors.
[0102] Chromosomal location, sequences, and annealing temperatures
(54-60.degree. C.) of the seven new primer pairs are as
follows:
4 for D8S3G7 (Chromosomal arm 8p): D8S3G7(F)
5'-GACCCTGTCAAGGAAAGAAAGAGA-3' (SEQ ID NO: 53) D8S3G7(R)
5'-CCATTTCAAATTTGGGACCACACT G-3' (SEQ ID NO: 54) for THO
(Chromosomal arm 11q): THO(F) 5'-AAGCTGCCCTAGTCAGCAC-3' (SEQ ID NO:
55) THO(R) 5'-GCTTCCGAGTGCAGGTCACA-3' (SEQ ID NO: 56) for D115488
(Chromosomal arm 11p): D115488(F) 5'-mGGAAGGAAGGAAGGAAAGG-3- ' (SEQ
ID NO: 57) D115488(R) 5'-CTGATAGCCTGACCTGACTGTG-3' (SEQ ID NO: 58)
for D135802 (Chromosomal arm 13q): D135802(F)
5'-CACAGTGAGACTCTATCTCAAA- AA-3' (SEQ ID NO: 59) D135802(R)
5'-TCAGACTGGCTTAGACTGTGG-3' (SEQ ID NO: 60) for D175695
(Chromosomal arm 17p): D175695(F) 5'-CTGGGCAACAAGAGCAAAATTC- -3'
(SEQ ID NO: 61) D175695(R) 5'-m GTTGTTGTTCATTGACTTCAGTCT-3' (SEQ ID
NO: 62) for D175654 (Chromosomal arm 17p): D175654(F)
5'-GACCTAGGCCATGTTCACAGCC- -3' (SEQ ID NO: 63) D175654(R)
5'-GACATCCATTGGCACCACCCCAA-3' (SEQ ID NO: 64) for D20548
(Chromosomal arm 20q): D20548(F) 5'-TCCAGTCCCATCTGGATTG-3'
D20548(R) 5'-GAAATAAGTGATGCTGTGATG-3'
[0103] The new markers were chosen by empirically screening 50
bladder tumors with 85 tri- and tetranucleotide microsatellite DNA
markers. Rates of loss of heterozygosity and shifts (new alleles)
were assessed and the best markers were included into this study.
Annealing temperatures, heterozygosity frequency, and length of
polymerase chain reaction products from each loci were obtained
from the Genome Data Base.
[0104] One primer of each marker pair was end-labeled with
[.gamma.-.sup.32P] ATP (Amersham, Arlington Heights, Ill.) using
T4-polynucleotide kinase (Gibco BRL, Gaithersburg, Md.). Genomic
DNA (50 ng) was subjected to 35 polymerase chain reaction cycles at
a denaturing temperature of 95.degree. C. for 30 sec, followed by
varying annealing temperatures ranging from 54-58.degree. C. for 1
min, an extension step at 70.degree. C. for 1 min and a final
extension step at 70.degree. C. for 5 min on Hybaid thermocyclers
(Hybaid, Teddington, UK). Polymerase chain reaction products were
then separated in denaturing 7% polyacrylamide-urea-formamide gels.
The running distances were calculated according to the expected
lengths of the polymerase chain reaction products. Autoradiography
was performed overnight at -80.degree. C. with Kodak X-OAAAT
scientific imaging film (Eastman Kodak, Rochester, N.Y.). Loss of
heterozygosity was scored in informative cases if a significant
reduction (>30%) in the ratio of the signals from the urine
and/or tumor alleles was observed in comparison with the
corresponding normal (germline) alleles in the adjacent lane.
[0105] The results of this Example show the feasibility of the
present invention. Loss of heterozygosity or mutations by
microsatellite DNA analysis (a positive test) was found in the
urine of eighteen (90%) of these patients. In each case, the
genetic change in urine DNA was identical to that identified in the
primary tumor. One of the two affected patients missed by
microsatellite DNA analysis displayed no mutations at any of the
twenty tested loci in the initial urine and tumor, but showed loss
of heterozygosity at two loci (D16S476 and D9S162) in the follow-up
urine sample collected five months preceding the eventual detection
and resection of a recurrent tumor by cystoscopy. The other patient
also showed no mutations in his initial urine or tumor sample with
this set of markers. Although both of these patients had
superficial, low grade tumors, similar tumors from other patients
demonstrated multiple genetic changes on several chromosomal
arms.
[0106] These patients were tested at routine cystoscopic evaluation
at approximately four to six month intervals for up to twenty-six
months. Recurrent tumors were observed in eleven of the twenty-one
patients. Ten of these eleven patients (91%) were diagnosed
correctly by microsatellite DNA analysis of the urine DNA collected
at the time of follow-up visits. The urine samples of affected
patients displayed multiple genetic changes on different
chromosomal arms, confirming a "positive" result at several markers
in many cases. These genetic changes were also identified in the
DNA of paired tumor samples from each case when available. In two
of these patients, a positive test preceded overt clinical
diagnosis by cystoscopy.
[0107] The single false-negative result was from a patient who had
a small pTa, GI recurrent tumor. Although urine samples and tumor
biopsies exhibited multiple DNA changes at the initial
presentation, the follow-up urine (and tumor) samples at five
months appeared to be free of mutations at the screened loci.
[0108] Recurrence-free status was correctly identified at follow-up
by microsatellite DNA analysis in ten of the ten patients (100%)
who were disease-free after cystoscopy. In nine of these ten cases,
genetic mutations present in urine at initial presentation reverted
to normal on follow-up analysis. One elderly patient underwent a
bladder-sparing treatment protocol for a T3 tumor. During
multimodality treatment (chemotherapy and radiation therapy), the
molecular test remained positive in the urine. At six month
follow-up, the patient demonstrated no evidence of recurrent
disease, and the molecular urine test reverted back to normal.
[0109] Cytologic analysis at the time of the last follow-up, also
performed in a blinded fashion, was available from seventeen
patients. Neoplastic cells were identified in only one of eight
patients (13%) with recurrent lesions (two of the patients with
recurrent disease were not evaluated by cytology). All nine
patients without recurrent disease were correctly identified as
negative by cytology (cytology was not performed in the two
remaining cases).
[0110] In this Example, ten of eleven patients with recurrent
transitional cell carcinoma of the urinary tract were identified by
microsatellite DNA analysis of the urine collected at the time of
routine follow-up. Even small tumors of low stage and grade
exhibited multiple genetic mutations, allowing precise and
definitive diagnosis. Most tumors were detected by loss of
heterozygosity; however, one patient was detected by identification
of "shifts" (new alleles) alone. The results of this Example
support the theory that malignant cells can undergo enrichment by
storage; tumor cells may be resistant to apoptosis and may survive
longer than normal cells. Moreover, because the tumor surfaces
contain rapidly growing cell populations, with mutations in
cell-cell adhesion, malignant tumor cells are shed more readily and
in greater numbers into the urine than normal cells.
[0111] The only patient (and the two patients at initial
presentation) whose recurrent tumor could not be diagnosed from the
urine had a small, low-grade lesion, without any microsatellite DNA
abnormalities at the twenty tested markers. Thus, the molecular
test did not miss any patients with genetic changes present at
these markers in the primary tumors. This suggests that the lack of
detection of loss of heterozygosity or shifts due to normal
(non-tumor) cell contamination has not been a cause of
false-negative results. Small Ta lesions show microsatellite DNA
mutations as often as more invasive lesions, but they often harbor
fewer regions of loss of heterozygosity. Theoretically, the use of
a higher number of markers might further increase the sensitivity
of the test.
[0112] Cytology detected one of eight patients with recurrent
disease and missed six of seven patients with pTa lesions. Cytology
usually detects approximately 50% of superficial lesions and in
positive cases can help establish the diagnosis before cystoscopy.
Failure to detect these small tumors may probably be less
significant clinically. Progression to muscle-invasive tumors
occurs in only 3% in this stage, and metastatic spread has been
observed in 5% of patients. However, patients with these small
lesions may also benefit from chemopreventive approaches to prevent
progression, in addition to definitive resection. In this regard,
molecular analysis appears more promising as a method for detecting
these early lesions for additional interventions.
[0113] Ten of the ten patients without evidence of recurrent
disease were diagnosed correctly by microsatellite DNA analysis of
the urine samples. Moreover, all examinations during clinically
confirmed disease-free intervals also served as negative controls
for each patient. Most patients with bladder cancer will develop
recurrence within two years, and thus longer follow-up will be
necessary in some patients to confirm the accuracy of molecular
diagnosis.
[0114] Two patients were positive several months before clinical
confirmation by cystoscopy. In these cases, the molecular changes
obviously preceded the clinically overt macroscopic findings seen
by cytoscopy. In another retrospective study in lung cancer, a
positive molecular test in the sputum of one patient was found
thirteen months before the development of lung cancer.
[0115] The status of recurrent disease was diagnosed correctly in a
patient, from the follow-up urine sample with marker D17S695. It
was intriguing that the recurrent tumor had three other distinct
molecular mutations, yet none of these could be detected in the
urine. This patient had multi focal disease. It is probable that
the biopsy sample contained a clonal neoplastic population
different from that identified in the urine. Biopsies from the
other lesions were not available to definitely ascertain which
tumor shed the predominant cell population into the urine.
[0116] The data in this Example provide an insight into the
potential usefulness of microsatellite DNA urine analysis for
monitoring patients for recurrent disease. This Example shows a
high sensitivity for this assay in detecting recurrent tumors,
demonstrating the first potential clinical utility for this
approach. Moreover, the use of additional markers capable of
identifying other areas of loss of heterozygosity may further
improve the sensitivity of this test for bladder cancer. In
addition to providing a positive or negative test, this assay may
provide abundant molecular information regarding tumor progression
and prognosis (see FIG. 5).
EXAMPLE 4
Analysis of Saliva in Cases of Head and Neck Cancer
[0117] One hundred and five microsatellite DNA markers were
screened in primary lung, and head and neck cancer, to find those
most amenable to microsatellite DNA shifts. The eight best markers
show the highest frequency of these shifts. These were tested in
twenty-one paired samples of tumor and saliva in patients with head
and neck cancer. The saliva samples were obtained by both swabbing
and rinsing the mouth of the affected patients. In summary, fifteen
out of the twenty-one cancer cases with just these eight markers
were detected. In eight of these cases, a new allele or shift was
identified in both a tumor and saliva, three patients in which
there was both loss of heterozygosity and instability in the saliva
and tumor, and four additional patients which showed only loss of
heterozygosity in both the tumor and in the saliva with these
markers. In addition, twenty-two control samples were tested of
patients without cancer and found none of these mutations in
saliva.
[0118] The invention now being fully described, it will be apparent
to one of ordinary skill in the art that many changes and
modifications can be made without departing from the spirit or
scope of the invention.
Sequence CWU 1
1
* * * * *