U.S. patent application number 09/863806 was filed with the patent office on 2002-12-26 for detection of neoplasia by analysis of saliva.
This patent application is currently assigned to Johns Hopkins University School of Medicine. Invention is credited to Sidransky, David.
Application Number | 20020197608 09/863806 |
Document ID | / |
Family ID | 21901047 |
Filed Date | 2002-12-26 |
United States Patent
Application |
20020197608 |
Kind Code |
A1 |
Sidransky, David |
December 26, 2002 |
Detection of neoplasia by analysis of saliva
Abstract
Methods for detection of a cell proliferative disorder, such as
cancer, are provided utilizing analysis of target mutant nucleic
acids in saliva specimens are described. The presence of target
mutant nucleic acids is indicative of a neoplastic disorder of the
lung or the head and neck.
Inventors: |
Sidransky, David;
(Baltimore, MD) |
Correspondence
Address: |
Lisa A. Haile, Ph.D.
Gray Cary Ware & Freidenrich LLP
Suite 1600
4365 Executive Drive
San Diego
CA
92121-2189
US
|
Assignee: |
Johns Hopkins University School of
Medicine
|
Family ID: |
21901047 |
Appl. No.: |
09/863806 |
Filed: |
May 22, 2001 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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09863806 |
May 22, 2001 |
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09038637 |
Mar 10, 1998 |
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09038637 |
Mar 10, 1998 |
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08579223 |
Dec 28, 1995 |
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08579223 |
Dec 28, 1995 |
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08152313 |
Nov 12, 1993 |
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Current U.S.
Class: |
435/6.16 |
Current CPC
Class: |
C12Q 2600/156 20130101;
C12Q 1/6886 20130101; C07K 14/4703 20130101; A61B 10/0051 20130101;
C07K 14/82 20130101; C12Q 1/6827 20130101 |
Class at
Publication: |
435/6 |
International
Class: |
C12Q 001/68 |
Claims
1. A method for detecting cancer in a subject comprising: isolating
nucleic acid from a saliva specimen taken from the subject, and
assaying the nucleic acid for a genetic mutation associated with
cancer, wherein the presence of the mutation indicative of cancer
in the subject.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] The present invention is a continuation-in-part of U.S. Pat.
No. 5,726,223, filed on Dec. 28, 1995 which is a divisional
application of U.S. Pat. No. 5,561,041, filed Nov. 12, 1993.
BACKGROUND OF THE INVENTION
[0002] 1. Field of the Invention
[0003] The present invention relates generally to detection of a
target nucleic acid sequence and specifically to early detection of
preneoplasia or cancer in a subject by analysis of target mutant
nucleic acid sequences in a saliva specimen from the subject.
[0004] 2. Description of Related Art
[0005] An increasing body of evidence implicates somatic mutations
as causally important in the induction of human cancers. These
somatic mutations may accumulate in the genomes of previously
normal cells, some of which may then demonstrate the phenotypes
associated with malignant growth. Such oncogenic mutations may
include a number of different types of alterations in DNA
structure, including deletions, translocations and single
nucleotide alterations. The latter, also known as point mutations,
may frequently be involved in carcinogenesis, in that a variety of
mutagenic chemicals induce such mutations. In addition, such
mutations may occur spontaneously as a result of mistakes in DNA
replication.
[0006] Advances in recombinant DNA technology have led to the
discovery of normal cellular genes (proto-oncogenes and tumor
suppressor genes) which control growth, development, and
differentiation. Under certain circumstances, regulation of these
genes is altered and cause normal cells to assume neoplastic growth
characteristics. There are over 40 known proto-oncogenes and
suppressor genes to date, which fall into various categories
depending on their functional characteristics. These include, (1)
growth factors and growth factor receptors, (2) messengers of
intracellular signal transduction pathways, for example, between
the cytoplasm and the nucleus, and (3) regulatory proteins
influencing gene expression and DNA replication.
[0007] Point mutations have been directly implicated in the
causation of many human tumors. Some tumors carry oncogenes of the
ras gene family, which differ from their normal cellular
counterpart proto-oncogenes by the presence of a point mutation at
one of a limited number of sites in these genes. Similarly, point
mutations in critical regions of tumor suppressor genes, such as
p53, are often detected in tumor cells. These mutations represent
qualitative changes in the tumor cell genome which distinguish
these cells from normal cells and provide a basis for diagnosis of
the genetic origin of a tumor under study. Identification of the
mutations that have created active oncogenes may provide important
diagnostic and prognostic clues for tumor development. For example,
a number of mutations have been found to alter the 12th codon of
the ras oncogenes, causing replacement of a normally present
glycine by any of a number of alternative amino acid residues. Such
amino acid substitutions create a potent transforming allele. Thus,
the presence of a particular nucleotide substitution may be a
strong determinant of the behavior of the tumor cell (e.g., its
rate of growth, invasiveness, etc.). As a result, DNA probes for
oncogene mutations have promise as diagnostic reagents in clinical
oncology.
[0008] Among the various types of neoplasms, a number of those
which are found in the lungs are associated with oncogenic
mutations. Lung cancer is the leading cause of cancer related
deaths in Western countries. The prognosis for patients with lung
cancer is primarily dependent on the stage of the tumor at the time
of clinical diagnosis. Currently, only 25 to 40 percent of all lung
tumors are considered resectable at the time of initial assessment.
Patients diagnosed early with stage I tumors have a 40-70% survival
following surgical resection. An attempt at lung cancer screening
through the use of tri-annual saliva cytology and annual chest
x-ray has proven inadequate for the early detection of lung cancer.
Alternatively, the finding that tumors progress through a series of
well-defined genetic changes, including point mutations in
oncogenes, has stimulated efforts to develop additional,
non-invasive tests that could more reliably detect neoplasms of the
lung. For example, U.S. Pat. Nos. 5,561,041 and 5,726,223 describe
detection of neoplasia of the lung by analyzing sputum samples.
These patents are herein incorporated by reference in their
entirety.
[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
polyinerase 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
(Peltomaki 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); Peltomaki et al., Cancer Res. 53: 5853
(1993); Gonzalez-Zulueta et al., Cancer Res. 53: 5620 (1993)).
[0014] Early detection of preneoplasia or cancer is crucial to
increase the probability for treating such disorders. A
non-invasive, simple screening test would provide health care
workers with a tool for performing early diagnostic or prognostic
screens.
SUMMARY OF THE INVENTION
[0015] The present invention arose from the unexpected finding that
nucleic acid having a mutant nucleotide sequence associated with
lung neoplasia or head and neck cancer is present in detectable
levels in saliva specimens from patients having or at risk of
having lung neoplasia or head and neck cancer.
[0016] As a consequence of this discovery, the present invention
represents a significant advance over such techniques as tissue
biopsy by providing a non-invasive, rapid, and accurate method for
detecting mutant nucleotide sequences associated with lung
neoplasia or neoplasia of the head and neck. The DNA amplification
based approach of the invention can identify one cell carrying a
mutant gene among a large excess (greater than 10,000) of normal
cells. Based on this finding, it is now possible to detect various
other target nucleic acids associated with other disease states.
The present invention provides a method which can be used to screen
high-risk populations to detect preneoplasia or cancer and to
monitor high risk patients undergoing chemoprevention or
chemotherapy.
[0017] In a first embodiment, the invention provides a method for
detecting a neoplastic disorder of the head and neck or lung in a
subject. The method includes providing a nucleic acid sample from a
saliva specimen from the subject; and detecting the presence of a
target mutant nucleotide sequence, wherein the presence of the
target nucleotide sequence in the saliva is indicative of a
neoplastic disorder of the head and neck or lung. Preferably, the
subject is a human.
BRIEF DESCRIPTION OF THE DRAWINGS
[0018] FIG. 1 is a table showing a series of microsatellite loci
for a number of patients.
[0019] FIG. 2 shows a graph of microsatellite markers having either
shifts or LOH.
[0020] FIG. 3 shows a graph of tumor detection in saliva by shift
versus LOH.
DETAILED DESCRIPTION OF THE INVENTION
[0021] The present invention relates to a method of detecting a
preneoplasia or neoplasia disorder or cancer in a subject by
analyzing nucleic acid having a mutant nucleotide sequence present
in saliva, wherein the presence of the mutant or altered nucleic
acid sequence is associated with neoplasia or preneoplasia of the
lung or head and neck.
[0022] In its broadest sense, the present invention allows the
detection of any neoplastic disorder of any organ for any target
nucleic acid sequence of diagnostic or therapeutic relevance, where
the target nucleic acid sequence is present in saliva. Thus, the
target nucleotide sequence may be, for example, a mutant
nucleotide, a restriction fragment length polymorphism (RFLP), a
nucleotide deletion, a nucleotide substitution, or any other
mammalian nucleic acid sequence of interest.
[0023] Further, essentially any type of allelic imbalance can be
detected in the saliva specimen. 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.
[0024] 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 or "loss of heterozygosity" (LOH). Methods of
detection of LOH are desribed in co-pending U.S. patent application
Ser. No. 08/968,733, which is herein incorporated by reference in
its entirety.
[0025] Mutations in target nucleic acid can be identified in the
method of the invention and used to screen for various cancers.
"Mutation" is the process whereby changes occur in the quantity or
structure of the genetic material of an organism. Mutations are
permanent alterations in the genetic material which may lead to
changes in phenotype. Mutation can involve modifications of the
nucleotide sequence of a single gene, blocks of genes or whole
chromosomes. Changes in single genes may be the consequence of
point mutations, which involve the removal, addition or
substitution of a single nucleotide base within a DNA sequence, or
they can be the consequence of changes involving the insertion or
deletion of large numbers of nucleotides. Modifications of whole
chromosomes include both changes in number or structural changes
involving chromosome abnormalities. Numerical chromosome mutations
can involve multiples of the complete karyotype, termed
"polyploidy", or they may involve deviations from the normal number
of chromosomes, termed "aneuploidy". Mutations can arise
spontaneously as a result of events such as errors in the fidelity
of DNA replication or the movement of transposable genetic elements
within genomes. They are also induced following exposure to
chemical or physical mutagens. Such mutation-inducing agents
include ionizing radiations, ultraviolet light and a diverse range
of chemicals such as the alkylating agents, and polycyclic aromatic
hydrocarbons all of which are capable of interacting either
directly or indirectly (generally following some metabolic
biotransformations) with nucleic acids. The DNA lesions induced by
such environmental agents may lead to modifications of the base
sequence when the affected DNA is replicated or repaired and thus
to a mutation.
[0026] An increasing body of evidence implicates somatic mutations
as causally important in the induction of human cancers. These
somatic mutations may accumulate in the genomes of previously
normal cells, some of which may then demonstrate the phenotypes
associated with malignant growth. Such oncogenic mutations may
include a number of different types of alterations in DNA
structure, including deletions, translocations and single
nucleotide alterations. The latter, also known as point mutations,
may frequently intervene in carcinogenesis, since a variety of
mutagenic chemicals induce such mutations. In addition, such
mutations may occur spontaneously as a result of mistakes in DNA
replication. As used herein the term "mutant or mutated" as applied
to a target neoplastic nucleotide sequence shall be understood to
include but not be limited to a mutation, a restriction fragment
length polymorphism, a nucleic acid deletion, or a nucleic acid
substitution and the like. A point mutation constitutes a single
base change in a DNA strand, for example a G residue altered to a
T. Such a mutation may alter the identity of the codon in which it
lies thereby creating a missense mutation or nonsense mutation.
Transition mutations involve the substitution of one purine in the
DNA by another purine or one pyrimidine by another pyrimidine, that
is A by G and vice versa, or T by C and vice versa. Transversions
involve the replacement of a purine by a pyrimidine and vice versa.
A missense mutation is a point mutation in which a codon is changed
into one encoding amino acid other than that normally found at a
particular position. A nonsense mutation is any mutation that
converts a codon specifying an amino acid into one coding for
termination of translation. A splicing mutation is any mutation
affecting gene expression by affecting correct RNA splicing.
Splicing mutations may be due to mutations at intron-exon
boundaries which alter splice sites. A polyadenylation site mutant
is a mutation of the consensus sequence required for addition of
poly(A) to the 3' end of mature mRNA and which results in premature
mRNA degradation. An insertion is any mutation caused by the
insertion of a nucleotide or stretch of nucleotides into a gene.
For example, naturally occurring insertion mutations can be the
result of the transposition of transposable genetic elements.
[0027] Mutations that occur in somatic cells are not transmitted to
the sexually produced offspring. However, such somatic mutations
may be transferred to descendant daughter cells and mutations in
some specific genes have been implicated in cancer. It is now clear
that mutations may lead to the induction of cancer when they occur
in one or more of a battery of normal genes referred to as the
proto-oncogenes. Proto-oncogenes may be modified by a variety of
mutational changes to produce the cancer-causing oncogenes of which
more than forty have been identified. Proto-oncogenes play an
essential part in the control of cell growth and differentiation
and disruption of their normal activity by mutational events may
lead to the aberrant growth characteristics observed in cancer
cells.
[0028] The existence of proto-oncogenes which can be converted or
activated to oncogenes by mutational events indicates a role for
exposure to environmental agents (radiation and chemicals) in
cancer induction. However, there are other types of genes, the
tumor suppressor genes or anti-oncogenes, which contribute to
cancer development only when they are inactivated or deleted. The
normal forms of such genes appear to suppress the development of
tumors whereas when only mutant forms are present tumors may
develop. Such genes were identified because most tumors that lack
functional copies of a suppressor gene (i.e., p53 or Rb) display
two identically mutated (i.e., homozygous) alleles, while the
unaffected tissues can be shown to carry one mutant tumor
suppressor allele and one wild-type allele. Therefore, the loss of
heterozygosity (LOH) at particular chromosome regions in tumor
cells (compared with somatic cells from the same individual) is
generally regarded as evidence for the unmasking of mutations in
tumor suppressor genes located in these regions.
[0029] In addition, changes in DNA sequence which do not result in
a detectable phenotypic change, termed "silent mutations", also
occur in structural genes. Mutations in structural genes are silent
if they do not change the amino acid inserted or if they result in
substitution of a residue that does not affect protein function.
Silent mutations can also be located outside protein-coding
regions. A neutral mutation constitutes a genetic change which
results in a phenotype which has no effect on an organism's
fitness. Thus, even when the substitution results in the insertion
of a different amino acid into a polypeptide, a missense mutation,
the amino acid may be an acceptable substitute and may not lead to
any significant change in the activity of the polypeptide. However,
some missense amino-acid changes can have drastic effects upon the
folding of polypeptide chains or upon the configuration of the
active site of an enzyme. Other base substitutions may have drastic
effects because they convert a triplet coding for an amino acid
into one of the three termination signals which lead to the
premature termination of polypeptide synthesis. Such nonsense
changes are usually accompanied by the loss of function of the gene
product.
[0030] When spontaneous or induced mutations are present in the
germ cells of sexually reproducing organisms they may be
transmitted to offspring. Such inherited mutations contribute to
the background mutational load where they contribute to disease and
disability. Inherited disease may be produced by both chromosome
abnormalities and by mutations in single genes, although the
influence of these two categories upon the observed mutation rate
will be uneven.
[0031] In addition to variations in nucleotide sequence due to
mutations, another major type of DNA polymorphism is a variation in
the number tandemly repeated sequences at a particular location in
the genome. The repeated sequence may be 10-60 nucleotides long, in
which case the tandem array is termed a "minisatellite". Groups of
shorter repeats (i.e., 2-5 nucleotides each) are termed
"microsatellites". Microsatellites, also known as simple sequence
repeats (SSRs) are highly variable polymorphic DNA sequences
composed of a variable number of tandemly arranged simple di-,
tri-, or tetranucleotide repeats. Generally, microsatellites are
not localized in any particular chromosomal region but are randomly
interspersed with other genomic DNA. Methods of detection of
microsatellite markers in cancer are described in U.S. patent
application Ser. No. 08/854,727, which is herein incorporated by
reference in its entirety.
[0032] In one embodiment, the method of the invention is applicable
for detection of mutant nucleotide sequences associated with benign
as well as malignant neoplasias (cancer). In a preferred embodiment
neoplasia of the lung or head and neck is detected, although the
method can be used to detect any neoplastic mutant nucleotide
sequence, regardless of origin, as long as the sequence is
detectably present in saliva. For example, head and neck cancers
shed cancer cells into saliva and can be detected. The term "head
and neck cancer", as used herein, encompasses any carcinoma in
tissues of the head and neck region of a subject. Such head and
neck carcinomas would include, for example, carcinoma of the mouth,
esophagus, throat, larynx, thyroid gland, tongue, lips, salivary
glands, nose, paranasal sinuses, nasopharynx, superior nasal vault
and sinus tumors, esthesioneuroblastoma, squamous call cancer,
malignant melanoma, sinonasal undifferentiated carcinoma (SNUC) or
blood neoplasia. Also included are carcinoma's of the regional
lymph nodes including cervical lymph nodes, prelaryngeal lymph
nodes, pulmonary juxtaesophageal lymph nodes and submandibular
lymph nodes (Harrison's Principles of Internal Medicine (eds.,
Isselbacher, et al., McGraw-Hill, Inc., 13th Edition, pp1850-1853,
1994).
[0033] Numerous nucleic acids having mutant nucleotide sequences
that produce an abnormal gene product are known to be associated
with various neoplasias. Among some of the most common mutant
nucleotide sequences are oncogenes and tumor suppressor genes, such
as the K-ras mutant oncogene, p16, deleted in colon cancer (DCC),
adenomatous polyposis coli (APC), familial adenomatous polyposis
coli (FAP) and p53. Of special significance in the present
invention is the detection of the K-ras mutant oncogene, p16, the
p53 tumor suppressor gene (Vogelstein, Nature, 348:681, 1990),
microsatellite loci, and LOH loci.
[0034] In order to analyze saliva specimens according to the method
of the invention, it may be desirable to enrich for epithelial
cells present in the specimen. This may be accomplished by mixing
the sample with an epithelial cell specific monoclonal antibody,
such as EBA-1 or Ber-Ep.sub.4 (Can. Res. 53:3455, 1993) available
from Dakopatts, Gestrop Denmark.) Other epithial cell specific
antibodies will be known to those of skill in the art.
[0035] When it is desired to amplify the mutant nucleotide sequence
before detection, this can be accomplished using oligonucleotide(s)
which are primers for amplification. These unique oligonucleotide
primers are based upon identification of the flanking regions
contiguous with the mutant nucleotide sequence and are capable of
substantially hybridizing with the flanking regions so that
amplification can proceed. For example, in the case of K-ras, these
oligonucleotide primers comprise sequences such as nucleotide
sequence 5'-AGGAATTCATGACTGAATATAAACTTGT-3'(SEQ. ID NO. 1) and/or
5'-ATCGAATTCTATGCATATTAAAACAAGATT-3' (SEQ. ID NO. 2) and sequences
complementary thereto. In the case of p53, the oligonucleotide
primers comprise sequences which are capable of hybridizing with
the flanking nucleotide sequence, wherein the primers are
[0036] 5'-GTAGGAATTCACTTGTGCCCTGACTT-3' (SEQ. ID NO. 3) and
[0037] 5'-CATCGAATTCCACTGACAACCACCCTT-3' (SEQ. ID NO. 4) (exons
5-6) and
[0038] 5'-GTAGGAATTCCAAGGCGCACTGGCCTC-3' (SEQ. ID NO. 5) and
[0039] 5'-ACTGAATTCTTCGTCTCCTCCACCGC-3' (SEQ. ID NO. 6) for exons
7-8, and
[0040] sequences complementary thereto.
[0041] The primers which can be used according to the method of the
invention embrace oligonucleotides of sufficient length and
appropriate sequence so as to provide specific initiation of
polymerization of a significant number of nucleic acid molecules
containing the target nucleic acid. In this manner, it is possible
to selectively amplify the specific target nucleic acid sequence
containing the nucleic acid of interest. Specifically, the term
"primer" as used herein refers to a sequence comprising two or more
deoxyribo-nucleotides or ribonucleotides, preferably at least
eight, which sequence is capable of initiating synthesis of a
primer extension product, which is substantially complementary to a
target nucleic acid strand. The oligonucleotide primer typically
contains 15-22 or more nucleotides, although it may contain fewer
nucleotides.
[0042] Experimental conditions conducive to synthesis include the
presence of nucleoside triphosphates and an agent for
polymerization, such as DNA polymerase, and a suitable temperature
and pH. The primer is preferably single stranded for maximum
efficiency in amplification but may be double-stranded. If
double-stranded, the primer is first treated to separate its
strands before being used to prepare extension products.
Preferably, the primer is an oligodeoxyribonucleotide. The primer
must be sufficiently long to prime the synthesis of extension
products in the presence of the inducing agent for polymerization.
The exact length of primer will depend on many factors, including
temperature, buffer, and nucleotide composition.
[0043] Primers used according to the method of the invention are
designed to be "substantially" complementary to each strand of
target nucleotide sequence to be amplified. Substantially
complementary means that the primers must be sufficiently
complementary to hybridize with their respective strands under
conditions which allow the agent for polymerization to function. In
other words, the primers should have sufficient complementarily
with the flanking sequences to hybridize therewith 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.
[0044] Oligonucleotide primers used according to the invention are
employed in any amplification process that produces increased
quantities of target nucleic acid. Typically, one primer is
complementary to the negative (-) strand of the mutant nucleotide
sequence and the other is complementary to the positive (+) strand.
Annealing the primers to denatured nucleic acid followed by
extension with an enzyme, such as the large fragment of DNA
Polymerase I (Klenow) or Taq DNA polymerase and nucleotides results
in newly synthesized + and -strands containing the target nucleic
acid. Because these newly synthesized nucleic acids are also
templates, repeated cycles of denaturing, primer annealing, and
extension results in exponential production of the region (i.e.,
the target mutant nucleotide sequence) defined by the primer. The
product of the amplification reaction is a discrete nucleic acid
duplex with termini corresponding to the ends of the specific
primers employed. Those of skill in the art will know of other
amplification methodologies which can also be utilized to increase
the copy number of target nucleic acid.
[0045] In nucleic acid hybridization reactions, the conditions used
to achieve a particular level of stringency will vary, depending on
the nature of the nucleic acids being hybridized. For example, the
length, degree of complementarity, nucleotide sequence composition
(e.g., GC v. AT content), and nucleic acid type (e.g., RNA v. DNA)
of the hybridizing regions of the nucleic acids can be considered
in selecting hybridization conditions. An additional consideration
is whether one of the nucleic acids is immobilized, for example, on
a filter.
[0046] An example of progressively higher stringency conditions is
as follows: 2.times. SSC/0.1% SDS at about room temperature
(hybridization conditions); 0.2.times. SSC/0.1% SDS at about room
temperature (low stringency conditions); 0.2.times. SSC/0.1% SDS at
about 42.degree. C. (moderate stringency conditions); and
0.1.times. SSC at about 68.degree. C. (high stringency conditions).
Washing can be carried out using only one of these conditions,
e.g., high stringency conditions, or each of the conditions can be
used, e.g., for 10-15 minutes each, in the order listed above,
repeating any or all of the steps listed. However, as mentioned
above, optimal conditions will vary, depending on the particular
hybridization reaction involved, and can be determined
empirically.
[0047] 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 Letters, 22:1859-1862, 1981). One
method for synthesizing oligonucleotides on a modified solid
support is described in U.S. Pat. No. 4,458,066. One method of
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.
[0048] Any saliva specimen nucleic acid, in purified or nonpurified
form, can be utilized 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, wherein DNA or RNA may be single stranded or double stranded.
If RNA is to be used as a template, enzymes, and/or conditions
optimal for reverse transcribing the template to DNA would be
utilized. In addition, a DNA-RNA hybrid which contains one strand
of each may be utilized. 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
utilized. The mutant nucleotide sequence to be amplified, may be a
fraction of a larger molecule or can be present initially as a
discrete molecule, so that the specific sequence constitutes 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.
[0049] Where the target mutant nucleotide sequence of the sample
contains two strands, it is necessary to separate the strands of
the nucleic acid before it can be used as the template. Strand
separation can be effected either as a separate step or
simultaneously with the synthesis of the primer extension products.
This strand separation can be accomplished using various suitable
denaturing conditions, including physical, chemical, or enzymatic
means; the word "denaturing" includes all such means. One physical
method of separating nucleic acid strands involves heating the
nucleic acid until it is denatured. Typical heat denaturation may
involve temperatures ranging from about 80.degree. to 105.degree.
C. for times ranging from about 1 to 10 minutes. Strand separation
may also be induced by an enzyme from the class of enzymes known as
helicases or by the enzyme RecA, which has helicase activity, and
in the presence of riboATP, is known to denature DNA. The reaction
conditions suitable for strand separation of nucleic acids with
helicases are described by Kuhn Hoffinann-Berling (CSH-Quantitative
Biology, 43:63, 1978) and techniques for using RecA are reviewed in
C. Radding (Ann. Rev. Genetics, 16:405-437, 1982).
[0050] If the nucleic acid containing the target nucleic acid to be
amplified is single stranded, its complement is synthesized by
adding one or two oligonucleotide primers. If a single primer is
utilized, a primer extension product is synthesized in the presence
of primer, an agent for polymerization, and the four nucleoside
triphosphates described below. The product will be complementary to
the single-stranded nucleic acid and will hybridize with a
single-stranded nucleic acid to form a duplex of unequal length
strands that may then be separated into single strands to produce
two single separated complementary strands. Alternatively, two
primers may be added to the single-stranded nucleic acid and the
reaction carried out as described.
[0051] When complementary strands of nucleic acid or acids are
separated, regardless of whether the nucleic acid was originally
double or single stranded, the separated strands are ready to be
used as a template for the synthesis of additional nucleic acid
strands. This synthesis is performed under conditions allowing
hybridization of primers to templates to occur. Generally synthesis
occurs in a buffered aqueous solution, preferably at a pH of 7-9,
most preferably about 8. Preferably, a molar excess (for genomic
nucleic acid, usually about 10.sup.8:1 primer:template) of the two
oligonucleotide primers is added to the buffer containing the
separated template strands. It is understood, however, that the
amount of complementary strand may not be known if the process of
the invention is used for diagnostic applications, so that the
amount of primer relative to the amount of complementary strand
cannot be determined with certainty. As a practical matter,
however, the amount of primer added will generally be in molar
excess over the amount of complementary strand (template) when the
sequence to be amplified is contained in a mixture of complicated
long-chain nucleic acid strands. A large molar excess is preferred
to improve the efficiency of the process.
[0052] In some amplification embodiments, the substrates, for
example, the deoxyribonucleotide triphosphates dATP, dCTP, dGTP,
and dTTP, are added to the synthesis mixture, either separately or
together with the primers, in adequate amounts and the resulting
solution is heated to about 90.degree.-100.degree. C. from about 1
to 10 minutes, preferably from 1 to 4 minutes. After this heating
period, the solution is allowed to cool to room temperature, which
is preferable for the primer hybridization. To the cooled mixture
is added an appropriate agent for effecting the primer extension
reaction (called herein "agent for polymerization"), and the
reaction is allowed to occur under conditions known in the art. The
agent for polymerization may also be added together with the other
reagents if it is heat stable. This synthesis (or amplification)
reaction may occur at room temperature up to a temperature above
which the agent for polymerization no longer functions. Thus, for
example, if DNA polymerase is used as the agent, the temperature is
generally no greater than about 40.degree. C. Most conveniently the
reaction occurs at room temperature.
[0053] The agent for polymerization may be any compound or system
which 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 which 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 which 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 agents for polymerization, however, which
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 which are described herein.
[0054] The newly synthesized mutant nucleotide strand and its
complementary nucleic acid strand will form a double-stranded
molecule under hybridizing conditions described above and this
hybrid is used in subsequent steps of the process. In the next
step, the newly synthesized double-stranded molecule is subjected
to denaturing conditions using any of the procedures described
above to provide single-stranded molecules.
[0055] The above process is repeated on the single-stranded
molecules. Additional agent for polymerization, nucleosides, and
primers may be added, if necessary, for the reaction to proceed
under the conditions prescribed above. Again, the synthesis will be
initiated at one end of each of the oligonucleotide primers and
will proceed along the single strands of the template to produce
additional nucleic acid. After this step, half of the extension
product will consist of the specific nucleic acid sequence bounded
by the two primers.
[0056] The steps of denaturing and extension product synthesis can
be repeated as often as needed to amplify the target mutant
nucleotide sequence to the extent necessary for detection. The
amount of the mutant nucleotide sequence produced will accumulate
in an exponential fashion.
[0057] The amplified product may be detected by analyzing it by
Southern blots without using radioactive probes. In such a process,
for example, a small sample of DNA containing a very low level of
mutant 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.
[0058] Nucleic acids having a mutation 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 PCR, 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). Thus, in a
preferred embodiment where the mutant nucleotide sequence to be
detected is K-ras, a hybridization probe is utilized which is
capable of hybridizing with mutant nucleotide sequences
comprising
1 5'-TTGCCTACGCCAACAGCTCC-3' (Val.sup.12) (SEQ. ID NO.7),
5'-TTGCCTACGCCATCAGCTCC-3' (Asp.sup.12) (SEQ. ID NO.8),
5'-TTGCCTACGCCACTAGCTCC-3' (Ser.sup.12) (SEQ. ID NO.9), or
5'-TTGCCTACGCCACAAGCTCC-3' (Cys.sup.12) (SEQ. ID NO.10)
[0059] complementary thereto. Where the mutant nucleotide sequence
to be detected is p53, a hybridization probe is utilized which is
capable of hybridizing with mutant nucleotide sequences
comprising
2 5'-CACAAACATGCACCTCAA-3' (His.sup.273) (SEQ. ID NO.11) or
5'-TGCGCCGGCCTCTCCCA-3' (Gly.sup.281) (SEQ. ID NO.12)
[0060] complementary thereto. The wild type K-ras and wild type p53
are detected by hybridizing with nucleotide probes which hybridize
with nucleotide sequences comprising
3 5'-TTGCCTACGCCACCAGCTCC-3' (SEQ. ID NO.13) and
5'-CCGGTTCATGGCGCCCAT-3' (SEQ. ID NO.14); respec- tively.
[0061] 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 thernocycler 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.
[0062] Exemplary target nucleotide sequences of the invention, to
which complementary oligonucleotide primers hybridize, include the
following:
4 SEQ ID NO:1 5'-CTTGTGTCCCGGCGTCTG-3' SEQ ID NO:2
5'-CAGCCCAGCAGGACCAGTA-3' SEQ ID NO:3 5'-TGGTAACAGTGGAATACTGAC-3'
SEQ ID NO:4 5'-ACTGATGCAAAAATCCTCAAC-3' SEQ ID NO:5
5'-GATGGGCAAACTGCAGGCCTGGGAAG-3' SEQ ID NO:6
5'-GCTACAAGGACCCTTCGAGCCCCGTTC-3' SEQ ID NO:7
5'-GATGGTGATGTGTTGAGACTGGTG-3' SEQ ID NO:8
5'-GAGCATTTCCCCACCCACTGGAGG-3' SEQ ID NO:9
5'-GTTCTGGATCACTTCGCGGA-3' SEQ ID NO:10 5'-TGAGGATGGTTCTCCCCAAG-3'
SEQ ID NO:11 5'-AGTGGTGAATTAGGGGTGTT-3' SEQ ID NO:12
5'-CTGCCATCTTGTGGAATCAT-3' SEQ ID NO:13 5'-CTGTGAGTTCAAAACCTATGG-3'
SEQ ID NO:14 5'-GTGTCAGAGGATCTGAGAAG-3' SEQ ID NO:15
5'-GCACGCTCTGGAACAGATTCTGGA-3' SEQ ID NO:16
5'-ATGAGGAACAGCAACCTTCACAGC-3' SEQ ID NO:17
5'-TCACTCTTGTCGCCCAGATT-3' SEQ ID NO:18 5'-TATAGCGGTAGGGGAGATGT-3'
SEQ ID NO:19 5'-TGCAAGGAGAAAGAGAGACTGA-3' SEQ ID NO:20
5'-AACAGGACCACAGGCTCCTA-3' SEQ ID NO:21
5'-TCTCTTTCTTTCCTTGACAGGGTC-3' SEQ ID NO:22
5'-CAGTGTGGTCCCAAATTTGAAATGG-3' SEQ ID NO:23
5'-GTGCTGACTAGGGCAGCTT-3' SEQ ID NO:24 5'-TGTGACCTGCACTCGGAAGC-3'
SEQ ID NO:25 5'-CCTTTCCTTCCTTCCTTCC-3' SEQ ID NO:26
5'-CACAGTCAGGTCAGGCTATCAG-3' SEQ ID NO:27
5'-TTTTTGAGATAGAGTCTCACTGTG-3' SEQ ID NO:28
5'-CCACAGTCTAAGCCAGTCTGA-3' SEQ ID NO:29
5'-GAATTTTGCTCTTGTTGCCCAG-3' SEQ ID NO:30
5'-AGACTGAAGTCAATGAACAACAAC-3' SEQ ID NO:31
5'-GGCTGTGAACATGGCCTAGGTC-3' SEQ ID NO:32
5'-TTGGGGTGGTGCCAATGGATGTC-3'
[0063] Other primers are listed below and the target sequences to
which they hybridize can be readily determined by those of skill in
the art.
[0064] Exemplary oligonucleotide primers, of the invention, include
the following but are not meant to be limiting in any way. Those of
skill in the art can identify other loci to amplify and similarly
design appropriate primers for amplification of nucleic acid from
saliva.
5 SEQ ID 5'-CAGACGCCGGGACACAAG-3' NO:33 SEQ ID
5'-TACTGGTCCTGCTGGGCTG-3' NO:34 SEQ ID D21S1245(F)
5'-GTCAGTATTACCCTGTTACCA-3' NO:35 SEQ ID D21S1245(R)
5'-GTTGAGGATTTTTGCATCAGT-3' NO:36 SEQ ID
5'-CTTCCCAGGCCTGCAGTTTGCCCATC-3' NO:37 SEQ ID
5'-GAACGGGGCTCGAAGGGTCCTTGTAGC-3' NO:38 SEQ ID DRPLA(F)
5'-CACCAGTCTCAACACATCACCATC-3' NO:39 SEQ ID DRPLA(R)
5'-CCTCCAGTGGGTGGGGAAATGCTC-3' NO:40 SEQ ID
5'-TCCGCGAAGTGATCCAGAAC-3' NO:41 SEQ ID 5'-CTTGGGGAGAACCATCCTCA-3'
NO:42 SEQ ID D14S50(F) 5'-AACACCCCTAATTCACCACT-3' NO:43 SEQ ID
D14S50(R) 5'-ATGATTCCACAAGATGGCAG-3' NO:44 SEQ ID FgA(F)
5'-CCATAGGTTTTGAACTCACAG-3' NO:45 SEQ ID FgA(R)
5'-CTTCTCAGATCCTCTGACAC-3' NO:46 SEQ ID D20548(F)
5'-TCCAGAATCTGTTCCAGAGCGTGC-3' NO:47 SEQ ID D20548(R)
5?-GCTGTGAAGGTTGCTGTTCCTCAT-3' NO:48 SEQ ID
5'-AATCTGGGCGACAAGAGTGA-3' NO:49 SEQ ID 5'-ACATCTCCCCTACCGCTATA-3'
NO:50 SEQ ID 5'-TCAGTCTCTCTTTCTCCTTGCA-3' NO:51 SEQ ID
5'-TAGGAGCCTGTGGTCCTGTT-3' NO:52 SEQ ID D8S3G7(F)
5'-GACCCTGTCAAGGAAAGAAAGAGA-3' NO:53 SEQ ID D8S3G7(R)
5'-CCATTTCAAATTTGGGACCACACTG-3' NO:54 SEQ ID THO(F)
5'-AAGCTGCCCTAGTCAGCAC-3' NO:55 SEQ ID THO(R)
5'-GCTTCCGAGTGCAGGTCACA-3' NO:56 SEQ ID D115488(F)
5'-mGGAAGGAAGGAAGGAAAGG-3' NO:57 SEQ ID D115488(R)
5'-CTGATAGCCTGACCTGACTGTG-3' NO:58 SEQ ID D135802(F)
5'-CACAGTGAGACTCTATCTCAAAAA-3' NO:59 SEQ ID D135802(R)
5'-TCAGACTGGCTTAGACTGTGG-3' NO:60 SEQ ID D175695(F)
5'-CTGGGCAACAAGAGCAAAATTC-3' NO:61 SEQ ID D175695(R)
5'-mGTTGTTGTTCATTGACTTCAGTCT-3' NO:62 SEQ ID D175654(F)
5'-GACCTAGGCCATGTTCACAGCC-3' NO:63 SEQ ID D175654(R)
5'-GACATCCATTGGCACCACCCCAA-3' NO:64
[0065]
6 D13S1286 Dinucleotide Repeat AFMc021xg5a GATCCACCAAAGCATATTATGA
(F) AFMc02lxg5m TTAAGCATCTTGAATTTTGCCT (R) (Dib. C Nature 380:152-4
1996) D13S1289 AFMc031zfla GTTTCTCCAGAACAGAACCAATAAG (F)
AFMc031zflm TAACCATGTAAGCCAGTCCC (R) (Dib. C Nature 380:152-4 1996)
D13S1560 1E5F GGCTGAGACATAAGACTCACTTGAAC 1E5R
TCCATGATGGCTAATGATACTGAG D9S753 (F) ACAGAGCAAGGTTGCCCAG (R)
TCCACTCAGCACCAACAGT FGA F 5'CCA TAG GTT TTG AAC TCA CAG FGA R 5'CTT
CTC AGA TCC TCT GAC AC D4S243 F 5'TCA GTC TCT CTT TCT CCT TGC A
D4S243 R 5'TAG GAG CCT GTG GTC CTG TT ACTBP2 F 5'AAT CTG GGC GAC
AAG AGT GA ACTBP2 R 5'ACA TCT CCC CTA CCG CTA TA D8S307 F 5'GAC CCT
GTC AAG GAA AGA AAG AGA D8S307 R 5'CCA TTT CAA ATT TGG GAC CAC ACT
G D9S162 F 5'GCA ATG ACC AGT TAA GGT TC D9S162 R 5'AAT TCC CAC AAC
AAA TCT CC TFN-A F 5'TGC GCG TTA AGT TAA TTG GTT TFN-A R 5'GTA AGG
TGG AAA CCC CCA CT D9S171 F 5'AGC TAA GTG AAC CTC ATC TCT GTC T
D9S171 R 5'ACC CTA GCA CTG ATG GTA TAG TCT D9S747 F 5'GCC ATT ATT
GAC TCT GGA AAA GAC D92747 R 5'CAG GCT CTC AAA ATA TGA ACA AAA T
THO F 5'CAG CTG CCC TAG TCA GCA C THO R 5'GCT TCC GAG TGC AGG TCA
CA AGG CAA TAG AGA CCC TGT G D11S488 F 5'AAG GAA GGA AGG AAG GAA
AGG D11S488 R 5'CTG ATA GCC TGA CCT GAC TGT G GAT GAT GAA TTG TTA
CTG AGA G D13S802 F 5'CAC AGT GAG ACT CTA TCT CAA AAA D13S802 R
5'TCA GAC TGG CTT AGA CTG TGG MJD F 5'CCA GTG ACT ACT TTG ATT CG
MJD R 5'TGG CCT TTC ACA TGG ATG TGA A D16S310 F 5'GGG CAA CAA GGA
GAG ACT CT D16S310 R 5'AAA AAA GGA CCT GCC TTT ATC C D16S476 F
5'TTG CAC TCC ACT CTG GGC A D16S476 R 5'TTG CCT TGG CTT TCT GTT GG
D17S695 F 5'CTG GGC AAC AAG AGC AAA ATT C D17S695 R 5'TTT GTT GTT
GTT CAT TGA CTT CAG TCT D17S654 F 5'GAC CTA GGC CAT GTT CAC AGC C
D17S654 R 5'GAC ATC CAT TGG CAC CAC CCC AA MBP F 5'GGA CCT CGT GAA
TTA CAA TC MBP R 5'ATT TAC CTA CCT GTT CAT CC D18S51 F 5'GAG CCA
TGT TCA TGC CCA CTG D18S51 R 5'CAA ACC CGA CTA ACC AGC AAC D20S82
5'GCC TTG ATC ACA CCA CTA CA D20S82 5'GTG GTC ACT AAA GTT TCT GCT
D20S85 5'ATT ACA GTG TGA GAC CCT G D20S85 5'GAG TAT CCA GAG AGC TAT
TA D20S94 5'TTG CTC CAA CCA GGA GGC A D20S94 5'GAA CCA AGG AAG TTG
TTC AAC D20S77 5'GCA GTG AGT TCA TAT GGC TA 5'GTT TCT CTG TTC AGC
ACT TC PPT 5'TGG CGA GAC TCC ATC AAA G 5'CCT TU AAG CTG CAA CAA TTT
C CSF1R.4 5'CAG GTT GCT AAC CAC CCT GT 5'GTG CAC ACT TGG ACA GCA TT
D14S125 5'CTG ACT CCA GAG CCT GGG 5'GGT TGA ATG TGG CGT GTT C
D14S50 5'AAC ACC CCT AAT TCA CCA CT 5'ATG ATT CCA CAA GAT GGC AG
FGA 5'CCA TAG GTT TTG AAC TCA CAG 5'CTT CTC AGA TCC TCT GAC AC
D9S252 5'ACA ATG AAC ATC CAT ATA CCC 5'ACC ATG ATT TGT CAA CTC CTA
D14S140 5'GCA TCT TGT TAG GGC AGC C 5'CCT GCA GAT TTT TGA TTT ACC
TBP 5'GAC CCC ACA GCC TAT TCA GA 5'TTG ACT GCT GAA CGG CTG CA APOB
5'GAA GAT TGC AGA GCT TTC TGC CAC 5'GTA AGT TCT CCT GGA GCA AGC TTC
ACPP 5'GAG GCT ACA GTA AGC CAA GAG TGC 5'GCC TTC CAA AAT GCA AGG
ATT ACA D8S306 5'GTT GAA CAG GAG CAG TGA GAG 5'AGA GAA AGA GAG ACA
GAA AGA GAG A L17686 5'GCA CCA ATG CTC CAG AAA TG 5'TCA TGG TGC CAT
GAT AGG AG D8S341 5'TAG GTA GTA AAC TTC ATA CAC G 5'CTC ATC TGC CTA
TAT CAC AGC Z35281 5'TGA TCT TCA AAG AGA GTT CCG 5'AGT TCG ATA GCA
CTC GCC GT G08270 5'TGG CGC TGA TGC TCC ACA TTC 5'CTG GCT GAC AGA
TAA AGC ACT G29028 5'GCA GTG AGC TGA GAT AAT GC 5'TCA CTA GCA GAT
GCG ATA ATG UT5307 5'GGA TAT AGC TGG CAA TGG C 5'TCG GAA TGC CTA
CTT CCC AG D9S242 5'GTG AGA GTT CCT TCT GGC 5'ACT CCA GTA CAA GAC
TCT G D9S758 5'AGC CTG GGT GAC AAA GCA AG 5'GCT CCA CAC CAC ACA CAT
G D9S778 5'AGA TCG CGC CAT TGT ACT C 5'AGC TCA GGG CAT TCT GTG AA
D9S769 5'GGA CTG CAT CTT AGG TGC TA 5'CAC CAA TGC AGT CTA GCC T
D9S737 5'ACG CTG GGC TAA TTC TTG 5'AGA GGC CAG GAG TTT GAG L17835
5'TTG CAC CAC TAT ACT CCA GC 5'TCA GTT TAA GGT TCT CAC CTC D17S846
5'TCC TTT GTT GCA GAT TTC TTC 5'TGC ATA CCT GTA CTA CTT CAG D11S488
5'AGG CAA TAG AGA CCC TGT G 5'GAT GAT GAA TTG TTA CTG AGA G D9S243
5'AAG GGC TGT CAT GTA GCA AG 5'CAA TCC AGG GAA GTT TCC AT D9S242
5'GTG AGA GTT CCT TCT GGC 5'ACT CCA GTA CAA GAC TCT G D9S759 5'TGG
CAG TGC AAG ACT CTT T 5'TGA GGA TTC GAG CAA TGC T X80250 5'TCA CCA
CTG CAC TCC AGC CT 5'GGA CTC ACC TTA TGC CAT GT X912531 5'GAG TGA
CAA ATT GAG ACC TGT 5'CGT CAG CCT AAG GTG GAC AT UT5320 5'ACC GAG
AGA CTC TTG CCT C 5'TTG AGA TGA CCC TGA GAG TG D11S554 5'AGC AGA
GCA AGA CTG TCT CA 5'GAC TCA AAC ACC TTC ATC CTA X91253 5'GAG TGA
CAA ATT GAG ACC TGT 5'CCT CAG CCT AAG GTG GAC AT D8S321 5'GAT GAA
AGA ATG ATA GAT TAC AG 5'ATC TTC TCA TGC CAT ATC TGC
[0066] 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: 1008-12 (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.
[0067] In an embodiment of the invention, purified nucleic acid
fragments containing intervening sequences or oligonucleotide
sequences of 10-50 base pairs are radioactively labelled. The
labelled preparations are used to probe nucleic acid from saliva by
the Southern hybridization technique. Nucleotide fragments from
saliva, before or after amplification, are separated into fragments
of different molecular masses by gel electrophoresis and
transferred to filters which bind nucleic acid. After exposure to
the labelled probe, which will hybridize to nucleotide fragments
containing target nucleic acid sequences, binding of the
radioactive probe to target nucleic acid fragments is identified by
autoradiography (see Genetic Engineering, 1, ed. Robert Williamson,
Academic Press, (1981), 72-81). Alternatively, nucleic acid from
saliva can be bound directly to filters to which the radioactive
probe selectively binds nucleic acids having the sequence of
interest specific sequences and the degree of binding is
quantitated by directly counting the radioactive emissions.
[0068] Where the target nucleic acid is not amplified, detection
using an appropriate hybridization probe may be performed directly
on the separated mammalian nucleic acid. In those instances where
the target nucleic acid is amplified, detection with the
appropriate hybridization probe would be performed after
amplification.
[0069] The probes of the present invention can be used for
examining the distribution of the specific fragments detected, as
well as the quantitative (relative) degree of binding of the probe
for determining the occurrence of specific strongly binding
(hybridizing) sequences, thus indicating the likelihood for an
individual to be at low risk or high risk for neoplastic disease,
such as a lung carcinoma. Further cloning allows specific
evaluation of the number of mutant nucleotides (i.e., mutant cells)
allowing a precise estimate of risk to develop neoplastic
disease.
[0070] For the most part, the probe will be labelled with an atom
or inorganic radical, most commonly using radionuclides, but also
perhaps heavy metals. Conveniently, a radioactive label may be
employed. Radioactive labels include .sup.32P, .sup.125I, .sup.3H,
.sup.14C, or the like. Any radioactive label may be employed which
provides for an adequate signal and has sufficient half-life. Other
labels include ligands, which can serve as a specific binding pair
member for a labelled ligand, and the like. A wide variety of
labels have been employed in immunoassays which can readily be
employed in the present assay. The choice of the label will be
governed by the effect of the label on the rate of hybridization
and binding of the probe to mutant nucleotide sequence. It will be
necessary that the label provide sufficient sensitivity to detect
the amount of mutant nucleotide sequence available for
hybridization. Other considerations will be ease of synthesis of
the probe, readily available instrumentation, ability to automate,
convenience, and the like.
[0071] The manner in which the label is bound to the probe will
vary depending upon the nature of the label. For a radioactive
label, a wide variety of techniques can be employed. Commonly
employed is nick translation with an a .sup.32P-dNTP or terminal
labeling with radioactive .sup.32P employing .sup.32.gamma. P-ATP
and T4 polynucleotide kinase. Alternatively, nucleotides can be
synthesized where one or more of the elements present are replaced
with a radioactive isotope, e.g., hydrogen with tritium. If
desired, complementary labelled strands can be used as probes to
enhance the concentration of hybridized label.
[0072] Where other radionuclide labels are involved, various
linking groups can be employed. A terminal hydroxyl can be
esterified, with inorganic acids, (e.g., .sup.32P phosphate), or
.sup.14C organic acids, or else esterified to provide linking
groups to the label. Alternatively, intermediate bases may be
substituted with activatable linking groups which can then be
linked to a label.
[0073] Enzymes of interest as reporter groups will primarily be
hydrolases, particularly esterases and glycosidases, or
oxidoreductases, particularly peroxidases. Fluorescent compounds
include fluorescein and its derivatives, rhodamine and its
derivatives, dansyl, u-mbelliferone, and so forth. Chemiluminescers
include luciferin, and 2, 3-dihydrophtha-lazinediones (e.g.,
luminol).
[0074] The probe can be employed for hybridizing to a nucleotide
sequence affixed to a water insoluble porous support. Depending
upon the source of the nucleic acid, the manner in which the
nucleic acid is affixed to the support may vary. Those of ordinary
skill in the art know, or can easily ascertain, different supports
which can be used in the method of the invention.
[0075] Any mammalian cells present in saliva are treated to
liberate their nucleic acid. The target sequences containing mutant
nucleotides are amplified by PCR or other aforementioned
techniques. The amplified nucleic acid from a saliva specimen is
spotted or spread onto a filter to provide a plurality of
individual portions. The filter is an inert porous solid support
(e.g., nitrocellulose). The lysing and denaturation of nucleic
acid, as well as the subsequent washings, can be achieved with an
appropriate solution for a sufficient time to lyse the cells and
denature the nucleic acid. For lysing, chemical lysing will
conveniently be employed, as described previously for the saliva
lysis buffer. Other denaturation agents include elevated
temperatures, organic reagents (e.g., alcohols, amides, amines,
ureas, phenols and sulfoxides) or certain inorganic ions (e.g.,
thiocyanate and perchlorate).
[0076] In a preferred embodiment, the amplified nucleic acid
containing the mutant nucleotide is be cloned into a vector (e.g.,
plasmid, cosmid, bacteriophage) utilizing the 5' restriction sites
contained within the amplification primers. Each clone contains one
copy of the amplified target sequence. The clone is transferred to
filters as described above, followed by denaturation. Hybridization
with an oligonucleotide/specific for the mutant nucleotide allows
the detection of one mutant nucleotide among 10,000 normal
nucleotides which differ at a single base pair.
[0077] After denaturation, the filter is washed in an aqueous
buffered solution, such as Tris, generally at a pH of about 6 to 8,
usually 7. One or more washings may be involved, conveniently using
the same procedure as employed for the lysing and denaturation.
After the lysing, denaturing, and washes have been accomplished,
the nucleic acid spotted filter is dried at an elevated
temperature, generally from about 50.degree. C. to 70.degree. C.
Under this procedure, the nucleic acid is fixed in position and can
be assayed with the probe when convenient.
[0078] Pre-hybridization may be accomplished by incubating the
filter at a mildly elevated temperature for a sufficient time with
the hybridization solution without the probe to thoroughly wet the
filter. Various hybridization solutions may be employed, comprising
from about 20% to 60% volume, preferably 30%, of an inert polar
organic solvent. A common hybridization solution employs about 50%
formamide, about 0.5 to 1M sodium chloride, about 0.05 to 0.1M
sodium citrate, about 0.05 to 0.2% sodium dodecylsulfate, and minor
amounts of EDTA, ficoll (about 300-500 kD), polyvinylpyrrolidone,
(about 250-500 kD) and serum albumin. Also included in the
hybridization solution will generally be from about 0.5 to 5 mg/ml
of sonicated denatured DNA (e.g., calf thymus or salmon sperm) and
optionally from about 0.5 to 2% wt/vol glycine. Other additives may
also be included, such as dextran sulfate of from about 100 to
1,000 kD and in an amount of from about 8 to 15 weight percent of
the hybridization solution.
[0079] The particular hybridization technique is not essential to
the invention. Other hybridization techniques are described by Gall
and Pardue, Proc. Natl. Acad. Sci. 63:378, 1969; and John, et al.,
Nature, 223:582, 1969. As improvements are made in hybridization
techniques they can readily be applied in the method of the
invention.
[0080] The amount of labelled probe which is present in the
hybridization solution will vary widely, depending upon the nature
of the label, the amount of the labelled probe which can reasonably
bind to the filter, and the stringency of the hybridization.
Generally, substantial excess over stoichiometric concentrations of
the probe will be employed to enhance the rate of binding of the
probe to the fixed target nucleic acid.
[0081] Various degrees of stringency of hybridization may be
employed. The more severe the conditions, the greater the
complementarily that is required for hybridization between the
probe and the single stranded target nucleic acid sequence for
duplex formation. Severity can be controlled by temperature, probe
concentration, probe length, ionic strength, time, and the like.
Conveniently, the stringency of hybridization is varied by changing
the polarity of the reactant solution by manipulating the
concentration of formamide in the range of 20% to 50%. Temperatures
employed will normally be in the range of about 20.degree. C. to
80.degree. C., usually 30.degree. C. to 75.degree. C. (see,
generally, Current Protocols in Molecular Biology, Ausubel, ed.,
Wiley & Sons, 1989).
[0082] After the filter has been contacted with a hybridization
solution at a moderate temperature for a period of time sufficient
to allow hybridization to occur, the filter is then introduced into
a second solution having analogous concentrations of sodium
chloride, sodium citrate and sodium dodecylsulfate as provided in
the hybridization solution. The time for which the filter is
maintained in the second solution may vary from five minutes to
three hours or more. The second solution determines the stringency,
dissolving cross duplexes and short complementary sequences. After
rinsing the filter at room temperature with dilute sodium
citrate-sodium chloride solution, the filter may now be assayed for
the presence of duplexes in accordance with the nature of the
label. Where the label is radioactive, the filter is dried and
exposed to X-ray film.
[0083] The label may also be labeled with a fluorescence moiety
which can then be probed with a specific antifluorescence antibody.
Conjugated to this antibody is horseradish peroxidase enzyme, for
example, able to catalyze a chemiluminescent reaction. Production
of light can then be seen on rapid exposure to film.
[0084] The materials for use in the assay of the invention are
ideally suited for the preparation of a kit. Such a kit may
comprise a carrier means being compartmentalized to receive in
close confinement one or more container means such as vials, tubes,
and the like, each of the container means comprising one of the
separate elements to be used in the method.
[0085] In general, one of the container means may comprise a
hybridization probe which is or can be detectably labelled. A
second container may comprise a saliva/epithelial cell lysis
buffer. The kit may also have containers containing nucleotide(s)
for amplification of the target nucleic acid sequence and/or a
container comprising a reporter-means, such as a biotin-binding
protein, such as avidin or streptavidin, bound to a reporter
molecule, such as an enzymatic, florescent, or radionuclide
label.
[0086] The method of the invention provides the basis for a kit
useful for the detection of a target mutant nucleic acid sequence
in a saliva specimen. The presence of a target mutant nucleic acid
sequence in the saliva is indicative of the presence of a
neoplastic disorder (e.g., of the head and neck or lung). The kit
includes a carrier means being compartmentalized to receive therein
one or more containers. For example, a first container contains a
nucleic acid hybridization probe which hybridizes to the target
nucleic acid. For example, the target nucleic acid to which the
probe hybridizes is selected from the group consisting of
7 5'TTGCCTACGCCAACAGCTCC-3' (SEQ. ID NO.7),
5'TTGCCTACGCCATCAGCTCC-3' (SEQ. ID NO.8), 5'TTGCCTACGCCACTAGCTCC-3'
(SEQ. ID NO.9), 5'TTGCCTACGCCACAAGCTCC-3' (SEQ. ID NO.10),
5'CACAAACATGCACCTCAA-3' (SEQ. ID NO.11), 5'TGCGCCGGCCTCTCCCA-3'
(SEQ. ID NO.12), 5'TTGCCCACGCCACCAGCTCC-3' (SEQ. ID NO.13), and
5'CCGGTTCATGGCGCCCAT-3' (SEQ. ID NO.14).
[0087] Other target nucleic acid sequences can be determined by
those of skill in the art and include, but are not limited to
sequences of tumor suppressor genes (e.g., p53, p16), oncogenes
(e.g., K-ras), microsatellite markers, and the like. In addition,
the kit may include a a second container containing a means for
detecting hybridization of the probe with the target nucleic acid.
Such reporter means include a biotin-binding protein, such as
avidin or streptavidin, bound to a reporter molecule, such as an
enzymatic, fluorescent, or radionuclide label. Other reporter means
and labels are well known in the art. The kit may also include an
amplification polymerase and deoxyribonucleotide(s). The kit may
further include nucleic acid amplification buffer. Preferably, the
reagent that modifies unmethylated cytosine is bisulfite. The kit
of the invention is intended to provide the reagents necessary to
perform nucleic acid hybridization analysis as described
herein.
[0088] Techniques for obtaining saliva are usually based on
collection by aspiration or absorption to material that is chewed
or placed somewhere in the mouth, or any method which results in
oral evacuation. Saliva acquisition can be accomplished by any
means which allows for the isolation of a sample from a subject
that results in a sufficient quantity of fluid being obtained for
testing. Such methods can include, for example, the use of a
sterile, absorbent cotton swab (e.g., with a serrated edge designed
for use in collecting body fluids or cells. The swab head can be
designed to fit into a micro-centrifuge tube or combined with a
mini-vial transport system. The device can further consist of a
plastic cylinder fitted at one end with a plug-gasket-filter
assembly. The filter typically has an appropriate pore size to
allow passage of a clean test fluid into a container while
particulates are eliminated. Preferably, the saliva sample is
collected in saline (e.g., about 0.9% NaCl solution).
[0089] Oral rinses and other methods of obtaining saliva can be
utilized in the methods of the invention. Various personel,
including dental personel and other medical or non-medical workers
may collect the saliva sample for further analysis as described
herein. Examples of saliva collection are described in Example 1
herein.
[0090] Omni-SAL is a painless, non-invasive saliva collection
system designed to maintain sample integrity. An alternative to
blood or urine, saliva may be collected at any location with
minimal training. The system consists of a sterile collection
device with patented volume adequacy indicator, and a transport
tube containing a stabilizing buffer.
[0091] The above disclosure generally describes the present
invention. A more complete understanding can be obtained by
reference to the following specific examples which are provided
herein for purposes of illustration only and are not intended to
limit the scope of the invention.
EXAMPLE 1
Saliva Collection Protocol
[0092] Saliva can be obtained from a subject by a number of ways
that will be apparent to those of skill in the art, including oral
rinses, swabs, suction, and the like. The following are intended to
be non-limiting examples of saliva extraction from a subject.
[0093] 1. Pre-operative oral rinse--The patient or control subject
gargles and rinses with 25 cc of sterile 0.9% NaCl for 15 seconds.
If sample must be collected intraoperatively, the saline is placed
in the oral cavity of the supine anethetized patient and collected
using a suction trap.
[0094] 2. Intraoperative tumor swab--The surgeon uses a sterile
cotton-tipped applicator or powder-free gloved finger to swab the
surface of the epithelial lesion. The exfoliated cells are
collected in 25 cc of sterile 0.9% NaCl. If the patient will not be
treated surgically, this specimen is collected in clinic.
[0095] 3. Intraoperative nonspecific oral cavity swab--The surgeon
uses a cotton-tipped applicator or powder-free gloved finger to
swab the oral cavity and oropharynx, including as much of the
hypopharynx as feasible. The exfoliated cells are collected in 25
cc of sterile 0.9% NaCl. If the patient will not be treated
surgically, this specimen is collected in clinic.
[0096] 4. The investigator places the rinse specimens in 50 ml
Sarstedt conical centifuge tubes and spins at 2500 rpm for 15
minutes. The supernatant is decanted off and the pellet is used for
DNA extraction.
EXAMPLE 2
[0097] DNA was isolated from the saliva samples by standard methods
such as those described in U.S. Pat. Nos. 5,561,041 and 5,726,223,
which methods are herein incorporated by reference.
[0098] Microsatellite analysis was performed by using standard PCR
techniques known in the art. An exemplary experiment is as
follows:
8 1. End labeling of the primer -1.5 ml tube (small) 10 .mu.l ddH2O
5 .mu.l primer (F or R) 2 .mu.l Buffer (T4 buffer) 1 .mu.l
T4-Kinase 2 .mu.l p32 .gamma. ATP Total volume 20 .mu.l Incubation
for 30 min. In 37.degree. C.
[0099] 2. In PCR room setup tubes/wells for PCR analysis (template
DNA) Dilute genomic DNA to a working concentration of 10 ng/.mu.l;
normal and tumor DNA for each sample to be analysed (Label) N and T
for each sample and place 2.5 .mu.l into corresponding
tubes/wells
[0100] 3. Prepare reaction mix for each PCR reaction corresponding
the number of reaction/wells Primers F and R diluted to 100
ng/.mu.l working stock/dilution
[0101] 4. Take 7.5 .mu.l of the reaction mix and add to the
template tube prepared in step 2, to total reaction volume of 10
.mu.l
[0102] 5. Add 1 drop of mineral oil on top of each tube
[0103] 6. Place tubes/wells in PCR block and perform PCR
reaction:
9 95.degree. C. for 1 min. optimized temp. 50-58.degree. C. for 1
min. 72.degree. C. for 1 min. 35 Cycles 72.degree. C. 1 Cycle
[0104] 7. Complete PCR reaction and dilute with 4 .mu.l
Stop-buffer
[0105] 8. Load 3 .mu.l of reaction mix onto 7M Formamide Denaturing
Gel and electrophorese for 2-5 hours corresponding to the size of
the product
[0106] 9. Transfer gel to 3 mm Whatman paper and place in
cassette.
[0107] 10. Place film onto a gel and develop after 8-10 hours at
-80.degree. C.
[0108] RESULTS (N=32)
[0109] 1. Study #1: Cases with tumor-specific microsatellite
alterations detected in all exfoliated epithelial cell samples
using a panel of 8 microsatellite markers: {fraction (14/18)}
(77%).
[0110] a. Cases detected by shift markers: {fraction (10/18)}
(55%).
[0111] b. Cases detected by LOH markers: {fraction (7/18)}
(38%).
[0112] c. Cases detected by both shift and LOH markers: {fraction
(3/14)} (21%).
[0113] 2. Study #2: Cases with tumor-specific microsatellite
alterations detected in all exfoliated epithelial cell samples
using a panel of 21 microsatellite markers: {fraction (12/14)}
(86%).
[0114] a. Cases detected by shift markers: {fraction (9/14)}
(64%)
[0115] b. Cases detected by LOH markers: {fraction (4/14)}
(29%).
[0116] C. Cases detected by both shift and LOH markers: {fraction
(2/14)} (14%).
[0117] 3. Detection rate combining the two pilot studies (n=32):
{fraction (26/32)} (81%).
Summary of Microsatellite Analysis
[0118] Microsatellite DNA markers have been commonly used to
accurately detect genetic alterations in primary tumors. Saliva
from 22 patients with known neoplastic lesions of the oral,
pharyngeal and laryngeal mucosa were analyzed molecularly by
microsatellite analysis using methods described herein. Using 8
informative tetra-nucleotide microsatellite markers, 15 of 22 (68%)
patients displayed microsatellite changes in saliva matching those
seen in the respective tumor. 4 of 15 (27%) patients with genetic
alterations in their saliva displayed loss of heterozygosity (LOH)
in saliva and tumor DNA, 8 patients displayed microsatellite
instability in saliva and tumor DNA and 3 patients displayed both
LOH and microsatellite instability. Saliva DNA of 15 normal
controls was analyzed by this molecular method and none were found
to harbor microsatellite instability. This data suggests that PCR
based microsatellite analysis using a panel of selected
oligonucleotide markers susceptible to shift alterations, is a very
sensitive method of detection and surveillance of neoplastic
lesions of the head and neck, e.g., oropharynx.
[0119] FIGS. 1-3 show the results of analysis of saliva from
patients and the identification of mutations in nucleic acid
obtained from saliva specimens. The identification of mutations in
the nucleic acid correlated with the identification of a tumor of
the head and neck or lung in the patients.
[0120] The foregoing is meant to illustrate, but not to limit, the
scope of the invention. Indeed, those of ordinary skill in the art
can readily envision and produce further embodiments, based on the
teachings herein, without undue experimentation.
Sequence CWU 1
1
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