U.S. patent application number 09/420433 was filed with the patent office on 2002-07-25 for nucleic acid mutation detection in histologic tissue.
Invention is credited to SIDRANSKY, DAVID.
Application Number | 20020098480 09/420433 |
Document ID | / |
Family ID | 22665246 |
Filed Date | 2002-07-25 |
United States Patent
Application |
20020098480 |
Kind Code |
A1 |
SIDRANSKY, DAVID |
July 25, 2002 |
NUCLEIC ACID MUTATION DETECTION IN HISTOLOGIC TISSUE
Abstract
Methods are provided for detection of target neoplastic nucleic
acids in a tissue specimen, including a tumor margin or lymph node,
and reagents therefor, wherein the nucleic acids are preferably
mutant tumor suppressor genes or proto oncogenes. Methods for
treatment of cell proliferative diseases utilizing ribozymes or
antisense oligonucleotides specific for the target mutant nucleic
acids and/or replacement wild type genes are also disclosed.
Inventors: |
SIDRANSKY, DAVID;
(BALTIMORE, MD) |
Correspondence
Address: |
Lisa A. Haile
Gray Cary Ware & Freidenrich LLP
4365 Executive Drive Suite 1600
San Diego
CA
92121-2189
US
|
Family ID: |
22665246 |
Appl. No.: |
09/420433 |
Filed: |
October 12, 1999 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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09420433 |
Oct 12, 1999 |
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08181664 |
Jan 14, 1994 |
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6025127 |
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Current U.S.
Class: |
435/6.12 ;
435/91.2; 536/23.1; 536/23.5 |
Current CPC
Class: |
C12Q 2600/156 20130101;
C12N 2799/027 20130101; C12Q 1/6886 20130101; C07K 14/4746
20130101; A61K 48/00 20130101; C12Q 1/6827 20130101; C07K 14/82
20130101 |
Class at
Publication: |
435/6 ; 536/23.1;
536/23.5; 435/91.2 |
International
Class: |
C12Q 001/68; C07H
021/02; C07H 021/04; C12P 019/34 |
Claims
1. A method for detecting the presence of a mammalian target
nucleic acid which contributes to the etiology of a neoplasm, in a
tissue specimen, wherein the specimen is external to a primary
neoplasm and the specimen does not exhibit morphological
characteristics indicative of neoplastic pathology, and the target
nucleic acid is present in the primary neoplasm and the specimen,
the specimen being selected from the group consisting of a tumor
margin, a regional lymph node, the method comprising extracting the
nucleic acid present in the specimen and detecting the presence of
the target nucleic acid.
2. The method of claim 1, wherein the nucleic acid is amplified
before detecting.
3. The method of claim 2, wherein the amplification is by means of
oligonucleotides that hybridize to the flanking regions of the
target nucleic acid.
4. The method of claim 1, wherein the target nucleic acid contains
a mutation, a restriction fragment length polymorphism, a nucleic
acid deletion, or a nucleic acid substitution, as compared with a
corresponding wild-type nucleic acid in the specimen.
5. The method of claim 1, wherein the target nucleic acid is
selected from the group consisting of an oncogene and a tumor
suppressor gene.
6. The method of claim 5, wherein the tumor suppressor gene is
selected from the group consisting of APC, DCC, NF1, NF2, Rb, RET,
VHL, WT-1 and p53.
7. The method of claim 1, wherein the neoplasm is of the head.
8. The method of claim 1, wherein the neoplasm is of the neck.
9. The method of claim 1, wherein the neoplasm is benign.
10. The method of claim 1, wherein the neoplasm is malignant.
11. The method of claim 2, wherein the amplified nucleic acid is
cloned before detecting.
12. A method for detecting metastases in a subject having an
excised tumor comprising: a) isolating tissue from a surgical
margin or lymph node adjacent to said excised tumor; b) applying to
said tissue an oligonucleotide that preferentially hybridizes to a
neoplastic nucleic acid, and c) detecting the presence of said
neoplastic nucleic acid, wherein the presence of said neoplastic
nucleic acid indicates metastases.
13. The method according to claim 12 wherein no more than an
average of about one out of every ten thousand cells of said tissue
have a neoplastic nucleic acid.
14. The method according to claim 12 wherein said tissue appears
normal under a microscope.
15. The method according to claim 12 wherein said neoplastic
nucleic acid is a mutated tumor suppressor gene.
16. The method according to claim 15 wherein said tumor suppressor
gene is the p53 gene.
17. The method according to claim 12 wherein said neoplastic acid
is an oncogene.
18. A method for detecting a mammalian target neoplastic nucleic
acid in a tissue specimen which is external to a primary neoplasm,
comprising extracting the nucleic acid present in the specimen and
detecting the presence of the target nucleic acid.
Description
BACKGROUND OF THE INVENTION
[0001] 1. Field of the Invention
[0002] This invention relates to a method of detecting a target
neoplastic nucleic acid in histologic tissue external to a primary
neoplasm, and reagents useful therein.
[0003] 2. Description of Related Art
[0004] 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.
[0005] Advances in recombinant DNA technology have led to the
discovery of normal cellular genes (proto-oncogenes and tumor
suppressor genes) that control growth, development, and
differentiation. Under certain circumstances, the regulation of
these genes is altered, causing normal cells to assume neoplastic
growth behavior. 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.
[0006] 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. Mutation of the p53
suppressor gene is the most common alteration seen in epithelial
tumors and, indeed, in all human tumors (Hollstein, M. et al.,
Science 253:49-53, 1991).
[0007] When a tumor suppressor gene, such as p53, becomes mutated,
cell proliferation accelerates in the absence of the suppressor. On
the other hand, mutations in proto-oncogenes that transform them to
active oncogenes, such as a mutant ras oncogene, produces cell
proliferation caused by presence of the mutant gene itself. These
mutations represent qualitative changes in the tumor cell genome
that distinguish these cells from normal cells and provide a basis
for diagnosis of the genetic origin of a tumor under study.
[0008] 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, nucleotide hybridization probes
of oncogene mutations have promise as diagnostic reagents in
clinical oncology.
[0009] Head and neck squamous carcinoma, commonly associated with
mutant p53, kills over 11,000 Americans each year, yet little is
known concerning the genetic events involved in progression of
these malignancies. A number of neoplasms found in the
gastrointestinal tract, especially colorectal cancer, are better
understood and commonly associated with oncogene mutations.
Colorectal cancer is the third most common malignancy in the world,
with 570,000 new cases expected each year. Treatment of all cancers
depends on the tumor stage as determined by clinical evaluation and
surgical resection. The standard technique for assessing the spread
of a tumor is surgical resection of a primary tumor followed by
careful review using light microscopy of surgical margins and other
tissue, including lymph nodes. Under existing procedure, the
adjacent tissue is stained by standard techniques and assessed
under light microscopy for the presence of tumor cells. Accurate
therapeutic staging assesses the extent of tumor spread locally as
well as the presence of regional metastases in more distant sites,
such as lymph nodes. Accurate histopathologic assessment is
critical since it provides important prognostic indicators that
determine the probability of survival for a given patient following
surgical resection of the primary tumor.
[0010] Despite many years of research and billions of dollars in
expenditures the long term survival of patients with malignancies
remains disappointedly low, even where no tumor cells were detected
in the tumor margins or more distant tissues. This inability to
more accurately stage such patients might be due to the limitation
inherent is the standard histopathologic methodology which is based
upon visual observation and morphologic assessment under light
microscopy of adjacent tissue and regional lymph nodes. Thus, a
method which uses a more precise technique capable of determining
spread of the disease at an earlier stage might provide a more
accurate indication of the extent of tumor metastases into adjacent
and regional tissues. The present invention provides such a
method.
SUMMARY OF THE INVENTION
[0011] The present invention arose from the unexpected finding that
nucleic acid having a mutant nucleotide sequence associated with a
primary tumor is detectable in the adjacent histopathologic
surgical margins and more distant tissues, such as regional lymph
nodes, which are apparently "normal" when examined by standard
histological techniques. The invention accomplishes this greatly
improved accuracy by application of precise molecular
techniques.
[0012] As a consequence of this discovery, the present invention
represents a significant advance over such standard medical
techniques as visual, light microscopy tissue biopsy and
morphologic assessment of such tissue, by providing a rapid, and
accurate molecular biologic method for detecting at the molecular
level mutant nucleotide sequences associated with a primary tumor.
The approach of the invention is based upon DNA amplification and
can identify as few as a single 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 target nucleic acids from
cells previously associated with a large number of disease states
which are present in tissue that appears normal.
[0013] The present invention provides a method which can be used as
an adjunct to cytopathology, to screen high-risk populations and to
monitor high risk patients undergoing chemoprevention or
chemotherapy.
DESCRIPTION OF THE DRAWINGS
[0014] FIG. 1 is a tumor map showing physical findings at initial
presentation. Right alveolar ridge (ALV) lesion--cross-hatching;
left retromolar trigone lesion (RMT)--stippling. Floor of mouth
(FOM) leukoplakia--open circle. Bilateral clinically apparent neck
masses are cross-hatched to indicate precise biological linkage to
ALV.
[0015] FIG. 2 is an autoradiograph of sequencing gel demonstrating
p3 mutation in DNA from ALV tumor samples. (n)=normal DNA from
peripheral blood lymphocytes; (p)=primary ALV tumor; (r)=recurrent
tumor. p and r contain the identical G.fwdarw.C transversion
(arrows) in codon 281. A,C,G,T indicate adenosine, cytosine,
guanine and thymidine.
[0016] FIG. 3 is a schematic representation of molecular analysis
of p53 mutations in tumor, margins, and nodes. In each sample, a
portion of the p53 gene was amplified, cloned, and probed with a
mutant-specific oligomer. Clones containing DNA with the ALV
"signature" mutation at codon 281 hybridized with the nucleotide
hybridization probe are visible as dots. Primary ALV tumor (T)
produces many hybridized plaques, and a significant number are
present in the histologically negative posterior margin (M.sub.2)
and bilateral histologically positive lymph nodes (L.sub.1 and
L.sub.3). Two other marginal samples (M.sub.1 and M.sub.3) and
histologically negative nodes (L.sub.2 and L.sub.4) were negative
by molecular analysis.
[0017] FIG. 4A shows a sequencing gel audioradiograph from 6
invasive tumor DNA samples with lanes grouped together for rapid
identification of novel bands upon visual comparison. Arrow points
to T.fwdarw.C mutation (codon 220) in lane 1 from the tumor of
Patient H17 (Table 4).
[0018] FIG. 4B shows the sequencing gel and the arrow points to
C.fwdarw.T mutation (codon 146) in the tumor DNA of Patient C2 with
CIS (Table 4).
[0019] FIG. 5 shows immunohistochemical staining of p53
demonstrating intense nuclear staining in the carcinomatous portion
of a carcinoma arising in an inverted papilloma from Patient C7
(Table 4). The inverted papilloma portion did not stain. The
staining was performed using the CM-1 polyclonal anti-p53 antibody
and the ABC VectaStain.RTM. kit (Vector Laboratories).
[0020] FIG. 6 shows a schematic illustration of the molecular
analysis and histopathologic assessment of the surgical margins of
head and neck squamous carcinoma patients with intended curative
resections.
[0021] FIG. 7 shows a molecular anlaysis of surgical margins and
lymph nodes. Autoradiographs of plaque lifts hybridized with
mutant-specific oligomers derived from each patient's tumor are
shown. Positive (specific) hybridizing clones (black dots) are
detected in surgical margins (M), in lymph nodes (L), and in the
primary tumor (T) as a positive control. Details of each patient in
FIGS. 7A, B, and C, and percentage of tumor cells in margins and
lymph nodes appear in Tables 7, 8, and 9.
[0022] FIG. 7A (patient 4), shows that tumor cells are identified
in one margin (M) and lymph nodes (L1, L4 and L6) with negative
hybridization (empty circles) in L2 and L3.
[0023] FIG. 7B (patient 9), shows that many tumor cells are present
in M4 and M5 and fewer tumor cells in M1 and M2, with margin M3
free of tumor cells.
[0024] FIG. 7C (patient 16), shows that micrometastases are seen in
all 4 lymph nodes (L1-L4) to varying degrees.
[0025] FIG. 8 shows a photomicrograph of histopathologic margins.
Hematoxylin and eosin staining of positive (FIG. 8A), suspicious
(FIG. 8B), and negative (FIG. 8C) surgical margins are
demonstrated. All of the above margins had tumor cells detected by
molecular analysis. The percentage of neoplastic cells was 10% in
FIG. 8A (M2 from patient 13), 5% in FIG. 8B (M4 from patient 9),
and 0.25% in FIG. 8C (M2 from patient 15). Details of each patient
in FIGS. 8A, 8B, and 8C, and percentage of tumor cells in margins
and lymph nodes appear in Tables 7, 8, and 9.
DETAILED DESCRIPTION OF THE INVENTION
[0026] The present invention relates to a method of detecting a
neoplastic nucleic acid having a mutant nucleotide sequence present
in a histopathologic tissue sample external to a primary neoplasm,
such as a tumor margin specimen, comprising isolating the nucleic
acid present in the specimen and detecting the presence of the
neoplastic target nucleic acid wherein the presence of the nucleic
acid sequence is known to be associated with neoplasia, such as,
neoplasia of the head or neck.
[0027] The term "neoplastic" nucleic acid refers to a nucleic acid
sequence which directly or indirectly is associated with or causes
a neoplasm. As used herein the term "tumor margin" 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 usually appears to be normal to the
naked eye. More particularly, as used herein, "margin" refers to
the edge, border or boundary of a tumor. The margin generally
extends from about 1 mm to about 4mm from the primary tumor but can
be greater depending upon the size of the primary solid tumor. The
term "regional lymph node" refers to lymphoid tissue forming
lymphoid organs or nodes which are in close proximity to the
primary tumor. For example, regional lymph nodes in the case of
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. The term
"external to a primary neoplasm" means that the specimen is taken
from a site other than directly from the primary neoplasm
itself.
[0028] In its broadest sense, the present invention allows the
detection of any neoplastic target nucleic acid sequence of
diagnostic or therapeutic relevance, where the target nucleic acid
sequence is present in a tissue sample such as that heretofore
subjected to histopathologic examination using techniques of light
microscopy, such as the margins of a primary tumor or a regional
lymph node. 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 in such tissue specimens. As used herein the term "mutant
or mutated" as applied to a target neoplastic nucleotide sequence
shall be understood to encompass a mutation, a restriction fragment
length polymorphism, a nucleic acid deletion, or a nucleic acid
substitution.
[0029] In one embodiment, the method of the invention is applicable
to detection of mutant nucleotide sequences associated with benign
as well as malignant neoplasias and tumors. In a preferred
embodiment, neoplasia of the head or 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 a histologic specimen. For example, neoplasia
of regional lymph nodes associated with a primary mammary tumor can
be detected utilizing the method of the invention. The specimen can
also be chyle or blood.
[0030] Numerous nucleic acids having mutant nucleotide sequences
that produce an abnormal gene product are known to be associated
with various neoplasias. Among the most common mutant nucleotide
sequences are those occurring in oncogenes and tumor suppressor
genes, such as mutations of p53 and K-ras. Of special significance
in the present invention is the detection of mutations of the p53
tumor suppressor gene (Vogelstein, Nature, 348:681, 1990).
[0031] Nearly 100 oncogenes have been identified. Though the number
of known tumor suppressor genes is far less, the number is growing
rapidly. Some of the known or candidate tumor suppressor genes and
the neoplasias with which they are associated (J. Marx, Science,
261:1385-1367, 1993) are shown in Table 1 below.
1 TABLE 1 GENE CANCER TYPE HEREDITARY SYNDROME APC Colon Carcinoma
Familiar adenomatous polyposis DCC Colon Carcinoma -- NF1
Neurofibromas Neurofibromatosis type 1 NF2 Schwannomas and
Neurofibromatosis meningiomas type 2 p53 50% of all cancers Rb
Retinoblastoma Retinoblastoma RET Thyroid carcinoma; Multiple
endocrine pheochromocytoma neoplasia type 2 VHL Kidney carcinoma
von Hippel-Lindau disease WT-1 Nephroblastoma Wilms tumor
[0032] When it is desired to amplify the target nucleotide sequence
before detection, such as a mutant nucleotide sequence, this can be
accomplished using oligonucleotide(s) that are primers for
amplification. These unique oligonucleotide primers are based upon
identification of the flanking regions contiguous with the mutant
nucleotide sequence. For example, in the case of p53, these
oligonucleotide primers comprise sequences which are capable of
hybridizing with nucleotide sequences flanking the loci of
mutations, such as the following p53 nucleotide sequences:
2 a) 5'-AAGTCAGGGCACAAGTGAATTCCTAC-3' (SEQUENCE ID NO. 1) and b)
5'-AAGGGTGGTTGTCAGTGGAATTCGATG-3' (SEQUENCE ID NO. 2) for exons
5-6; c) 5'-GAGGCCAGTGCGCCTTGGAATTCCTAC-3' (SEQUENCE ID NO. 3) and
d) 5'-GCGGTGGAGGAGACGAAGAATTCAGT-3' (SEQUENCE ID NO. 4) for exons
7-8. e) sequences complementary to a.) through d.).
[0033] Primers that hybridize to these flanking sequences are, for
example, the following:
3 a) 5'-TTCACTTGTGCCCTGACTT-3' (SEQUENCE ID NO. 5); b)
5'-CTGGAAACTTTCCACTTGAT-3' (SEQUENCE ID NO. 6): c)
5'-CCACTGACAACCACCCTT-3' (SEQUENCE ID NO. 7); d)
5'-CCAAGGCGCACTGGCCTC-3' (SEQUENCE ID NO. 8); and e) sequences
complementary to a.) through d.).
[0034] One skilled in the art will be able to generate primers
suitable for amplifying target sequences of additional genes, such
as those flanking loci of known mutations in proto-oncogenes and
tumor suppressor genes, using routine skills known in the art and
the teachings of this invention.
[0035] In general, the primers used according to the method of the
invention embrace oligonucleotides of sufficient length and
appropriate sequence which provides 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.
Specifically, the term "primer" as used herein refers to a sequence
comprising 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 15-22 or more nucleotides, although it
may contain fewer nucleotides as long as the primer is of
sufficient specificity to allow essentially only the amplification
of the specifically desired target nucleotide sequence (i.e., the
primer is substantially complementary).
[0036] 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.
[0037] Primers used according to the method of the invention are
designed to be "substantially" complementary to each strand of
mutant 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 mutant nucleotide sequence. Preferably, the 3'
terminus of the primer that is extended has perfectly base paired
complementarity with the complementary flanking strand.
[0038] 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 or
ligases, 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.
[0039] 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.
[0040] The nucleic acid from any histologic tissue specimen, 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 (mRNA), wherein DNA or RNA may be
single stranded or double stranded. In the event that 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, such 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.
[0041] Where the target neoplastic 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 which is known to denature DNA. The
reaction conditions suitable for strand separation of nucleic acids
with helicases are described by Kuhn Hoffmann-Berling
(CSH-Quantitative Biology, 43:63, 1978) and techniques for using
RecA are reviewed in C. Radding (Ann. Rev. Genetics, 16:405-437,
1982).
[0042] 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.
[0043] 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. 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.
[0044] 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. 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.
[0045] 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.
[0046] 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.
[0047] 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.
[0048] The amplified 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
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.
[0049] 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 a p53 mutation, a hybridization probe is utilized which
is capable of hybridizing with mutant nucleotide sequences
comprising:
4 a) 5'-CACAAACATGCACCTCAA-3' (His.sup.273) (SEQUENCE ID NO. 9); b)
5'-TCTCCCAGTACAGGCACA-3' (Thr.sup.278) (SEQUENCE ID NO. 10); or c)
5'-TGCGCCGGCCTCTCCCA-3' (Gly.sup.281) (SEQUENCE ID NO. 11); and d)
sequences complementary to a.) through c.)
[0050] d) sequences complementary to a.) through c.).
[0051] In addition, hybridization probes described in Table 1 are
utilized to detect the listed mutant p53 sequences. The wild type
p53 is generally detected by hybridizing with a nucleotide probe
that hybridizes with a nucleotide sequence comprising
5'-CCGGTTCATGGCGCCCAT-3' (SEQUENCE ID NO.12), although any probes
can be utilized that hybridize with any p53 nucleotide sequence
that is not subject to mutation.
[0052] The invention also provides nucleotide sequence mutations
associated with a neoplasm, wherein the mutation is present at p53
codons 180, 187, 193, and 306. These codon orientations are
according to Matlashewski G., et al., Embo Journal.,
3:(13):3257-62, 1984 and Lamb, P., et al., Molecular & Cellular
Biology, 6(5):1379-85, 1986. The nucleotide sequences of the
mutations include: codon 180, GAG to TAG; codon 187, GGT to GAT;
codon 193, CAT to CGT; and 306, CGT to TGA. Other mutations at
these codons are also included. In accordance with the present
invention, these mutations are generally found in head and neck
tumors, although they may be present in other neoplastic cells as
well.
[0053] 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 a
histologic specimen by the Southern hybridization technique.
Nucleotide fragments from a histologic specimen, before or after
amplification, are separated into fragments of different molecular
masses by gel electrophoresis and transferred to filters that 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 the specimen can be bound
directly to filters to which the radioactive probe selectively
attaches by binding nucleic acids having the sequence of interest.
Specific sequences and the degree of binding is quantitated by
directly counting the radioactive emissions.
[0054] 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.
[0055] 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 head and neck squamous cell carcinoma.
[0056] For the most part, the probe will be detectably labelled
with an atom or inorganic radical, most commonly using
radionuclides, but also heavy metals can be used. Conveniently, a
radioactive label may be employed. Radioactive labels include
.sup.32P, .sup.125I, .sup.3H, .sup.14C, .sup.111In, .sup.99mTc, 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
routinely employed in immunoassays 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.
[0057] Other considerations will be ease of synthesis of the probe,
readily available instrumentation, ability to automate,
convenience, and the like.
[0058] 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
phosphate hydrolysis with alkaline phosphatase followed by labeling
with radioactive .sup.32P employing .sup.32P-NTP 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.
[0059] Where other radionucleotide 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 that can then be linked
to a label.
[0060] 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, umbelliferone, and so forth. Chemiluminescers
include luciferin, and 2,3-dihydrophthalazinediones (e.g.,
luminol).
[0061] 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
that can be used in the method of the invention.
[0062] The nucleic acid from a histologic specimen is cloned and
then spotted or spread onto a filter to provide a plurality of
individual portions (plaques). The filter is an inert porous solid
support, e.g., nitrocellulose. Any cells (or phage) present in the
specimen are treated to liberate their nucleic acid. 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 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.
[0063] 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.
[0064] Pre-hybridization may be accomplished by incubating the
filter with the hybridization solution without the probe at a
mildly elevated temperature for a sufficient time 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
of 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.
[0065] 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.
[0066] The amount of labelled probe present in the hybridization
solution will vary widely, depending upon the nature of the label,
the amount of the labelled probe that 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.
[0067] 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).
[0068] 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 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.
[0069] The label may also comprise a fluorescent moiety that can
then be probed with a specific antifluorescent antibody. For
example, horseradish peroxidase enzyme can be conjugated to this
antibody to catalyze a chemiluminescent reaction. Production of
light can then be seen on rapid exposure to film.
[0070] The present invention identifies mutations in a target
sequence, such as p53, that are unique to the primary tumor
isolated from a subject and metastatic sites derived from the
primary tumor. In the tumor cells, the mutated nucleotide sequence
is expressed in an altered manner as compared to expression in a
normal cell; therefore, it is possible to design appropriate
therapeutic or diagnostic techniques directed to this specific
sequence. Thus, where a cell-proliferative disorder is associated
with the expression of a particular mutated proto-oncogene or tumor
suppressor gene nucleic acid sequence, a nucleotide sequence that
interferes with the specific expression of the mutated gene at the
transcriptional or translational level can be used. This approach
utilizes, for example, antisense oligonucleotides and/or ribozymes
to block transcription or translation of a specific mutated mRNA,
either by masking that mRNA with an antisense nucleic acid or by
cleaving it with a ribozyme.
[0071] Antisense nucleic acids are DNA or RNA molecules that are
complementary to at least a portion of a specific mRNA molecule
(Weintraub, Scientific American, 262:40, 1990). To date, several
tumor suppressor genes and oncogenes have been targeted for
suppression or down-regulation including, but not limited to, p53
(V. S. Prasolov et al., Mol. Biol.
[0072] (Moscow) 22:1105-1112, 1988); ras (S. K. Anderson et al.,
Mol. Immunol. 26:985-991, 1989; D. Brown et al., Oncogene Res.
4:243-249, 1989); fos (B. Levi et al., Cell. Differ. Dev. 25
(Suppl):95-102, 1988; D. Mercola et al., Gene 72:253-265, 1988);
and myc (S. O. Freytag, Mol. Cell. Biol. 8:1614-1624, 1988; E. V.
Prochownik et al., Mol. Cell. Biol. 8:3683-3695, 1988; S. L. Loke
et al., Curr. Top. Microbiol. Immunol. 141:282-288, 1988).
[0073] It is not sufficient in all cases to block production of the
target mutant gene. As described in A. J. Levine, et al.,
(Biochimica et Biophisica Acta., 1032:119-136, 1990), there are at
least five types of mutations that can contribute to the tumor
phenotype. Briefly, Type I mutations are those mutations in genes
that result in abnormal protein products, which act in a positive
dominant fashion. Examples of such mutations are those in H-ras and
K-ras genes that result in amino acid changes at positions 12 or 61
in the protein, leading to a protein that binds GTP and is
constantly signaling for cell growth. Type II mutations are those
that result in overproduction of an oncoprotein, such as the
bcr-abl translocation that results in overproduction of a normal
myc protein and an altered abl protein. Type III mutations are loss
of function mutations wherein tumors arise as the result of loss of
both alleles, such as with the retinoblastoma sensitivity gene (Rb)
on human chromosome 13q14 and the Wilm's tumor sensitivity gene
localized at 11q3. In 75% of colorectal carcinomas, one allele at
the p1 2-p13.3 locus of chromosome 17 containing the p53 gene is
commonly deleted, and in some cases the other p53 allele which
remains in the colorectal cancer cells has been shown to produce a
mutant p53 protein that presumably contributes to tumorigenesis.
Type IV mutations are those that result in expression of a protein
that does not directly contribute to the growth of cells, but
enhances the ability of cancer cells to survive. For instance,
mutations to the v-erb-A gene results in erythoblasts transformed
with the altered gene being kept in the replication cycle. Type V
mutations result from addition of new genetic information into
tumor cells, commonly by way of a virus. In some cases the virus
integrates its DNA into the cellular genome to produce proteins
that bind to cellular negative regulators of growth, such as RB and
p53, and thus, in effect, mimic the Type III loss of function
mutation mechanism.
[0074] Antisense therapy can be used to block production of mutant
proteins that act directly to increase the probability of producing
neoplastic cells, such as in mechanism Type III, Type IV and Type V
mutations that mimic Type III. Antisense is also therapeutically
effective when mutation is not dominant, for instance when a
non-mutant allele remains that encodes the proper protein. However,
when the mutation is dominant, as in Type I mutations, and in cases
wherein either both alleles are deleted or one is deleted and the
other is mutant, as in certain Type III mutations, antisense
therapy is preferably accompanied by replacement therapy. In
replacement therapy a wild type gene is introduced into the target
cells identified as having a mutant tumor suppressor gene or
protooncogene which results in production of the wild type protein
necessary to forestall development of the neoplasia associated with
the identified mutant gene(s).
[0075] In the case of tumor suppressor genes, it is known that
introducing a suppressor gene into cultured cells either causes
cell death or causes no discernible changes, however, the cells may
no longer be tumorigenic in animals. Thus, in cases where ribozyme
and/or antisense therapy is accompanied by gene replacement
therapy, the chances are increased that the cell population
containing the mutant gene for which the ribozyme or antisense
oligonucleotide is specific will no longer contribute to
development of neoplasia in the subject being treated.
[0076] Synthetic antisense oligonucleotides are generally between
15 and 25 bases in length. Assuming random organization of the
human genome, statistics suggest that a 17-mer defines a unique
sequence in the cellular mRNA in human DNA; a 1 5-mer defines a
unique sequence in the cellular mRNA component. Thus, substantial
specificity for a selected genetic target is easily obtained using
the synthetic oligomers of this invention.
[0077] In the cell, the antisense nucleic acids hybridize to the
corresponding mRNA, forming a double-stranded molecule. The
antisense nucleic acids, interfere with the translation of the
mRNA, since the cell will not translate a mRNA that is
double-stranded. Antisense oligomers of about 15 nucleotides are
preferred, since they are easily synthesized and are less likely to
cause problems than larger molecules when introduced into the
target nucleotide mutant producing cell. The use of antisense
methods to inhibit the in vitro translation of genes is well known
in the art (Marcus-Sakura, Anal. Biochem., 172:289, 1988). Less
commonly, antisense molecules which bind directly to the DNA may be
used. Ribozymes are RNA molecules possessing the ability to
specifically cleave other single-stranded RNA in a manner analogous
to DNA restriction endonucleases. Through the modification of
nucleotide sequences that encode these RNAs, it is possible to
engineer molecules that recognize specific nucleotide sequences
associated with production of a mutated proto oncogene or tumor
suppressor gene in an RNA molecule and cleave it (Cech, J. Amer.
Med. Assn., 260:3030, 1988). A major advantage of this approach is
that, because they are sequence-specific, only target mRNAs with
particular mutant sequences are inactivated.
[0078] There are two basic types of ribozymes, namely,
tetrahymena-type (Hasselhoff, Nature, 334:585, 1988) and
"hammerhead"-type. Tetrahymena-type ribozymes recognize sequences
which are four bases in length, while "hammerhead"-type ribozymes
recognize base sequences 11-18 bases in length. The longer the
recognition sequence, the greater the likelihood that the sequence
will occur exclusively in the target mRNA species. Consequently,
hammerhead-type ribozymes are preferable to tetrahymena-type
ribozymes for inactivating a specific mRNA species, and 18-based
recognition sequences are preferable to shorter recognition
sequences.
[0079] Unmodified oligodeoxyribonucleotides are readily degraded by
serum and cellular nucleases. Therefore, as is well known in the
art, certain modifications of the phosphate backbone have conferred
nuclease resistance to antisense DNA. For instance
phosphorothioate, methylphosphonate, and .alpha.-anomeric
sugar-phosphate, backbone-modified oligomers have increased
resistance to serum and cellular nucleases. In addition,
methylphosphonates are nonionic and offer increased lipophilicity
to improve uptake through cellular membranes. The use of modified
oligonucleotides as antisense agents may require slightly longer or
shorter sequences because chemical changes in molecular structure
can affect hybridization (L. A. Chrisey et al., BioPharm 4:36-42,
1991). These backbone-modified oligos bind to a target sequence and
exert their inhibitory effects by blocking the binding of the
cell's translational machinery to a specific RNA or by inducing
ribonuclease H activity through the formation of RNA/DNA duplex
structures.
[0080] The present invention also provides gene therapy for the
treatment of cell proliferative or immunologic disorders that are
mediated by a mutated proto-oncogene or tumor suppressor gene, such
as a mutated p53 gene sequence. Such therapy would achieve its
effect by introduction of the specific antisense polynucleotide
and/or replacement wild type gene into cells identified by the
methods of this invention as having the proliferative disorder
caused by mutated genes. Whether the cell will require replacement
of the wild type gene encoding the tumor suppressor gene or
proto-oncogene as well as antisense therapy to prevent replication
of the mutant gene must be determined on a case by case basis and
will depend upon whether the mutation has a dominant effect, i.e.,
whether both alleles of the wild type gene have been destroyed so
that total absence of the gene has a cell proliferative effect.
[0081] Delivery of antisense proto-oncogene or tumor suppressor
polynucleotides specific for mutated genes as well as of
replacement wild type genes can be achieved using a recombinant
expression vector such as a chimeric virus or a colloidal
dispersion system. Preferred for therapeutic delivery of antisense
sequences is the use of liposomes, especially targeted
liposomes.
[0082] Various viral vectors that can be utilized for gene therapy
as taught herein include adenovirus, herpes virus, vaccinia, or,
preferably, an RNA virus such as a retrovirus. Preferably, the
retroviral vector is a derivative of a murine or avian retrovirus.
Examples of retroviral vectors in which a single foreign gene can
be inserted include, but are not limited to: Moloney murine
leukemia virus (MoMuLV), Harvey murine sarcoma virus (HaMuSV),
murine mammary tumor virus (MuMTV), and Rous Sarcoma Virus (RSV). A
number of additional retroviral vectors can incorporate multiple
genes. All of these vectors can transfer or incorporate a gene for
a selectable marker so that transduced cells can be identified and
generated. By inserting one or more sequences of interest into the
viral vector, along with another gene which encodes the ligand for
a receptor on a specific target cell, for example, the vector is
now target specific. Retroviral vectors can be made target specific
by inserting, for example, a polynucleotide encoding a sugar, a
glycolipid, or a protein. Preferred targeting is accomplished by
using an antibody to target the retroviral vector. Those of skill
in the art will know of, or can readily ascertain without undue
experimentation, specific polynucleotide sequences which can be
inserted into the retroviral genome to allow target specific
delivery of the retroviral vector containing the proto oncogene or
tumor suppressor antisense polynucleotide (i.e., a p 53 antisense
polynucleotide). A separate vector can be utilized for targeted
delivery of a replacement gene to the cell(s), if needed, or the
antisense oligonucleotide and the replacement gene can optionally
be delivered via the same vector since the antisense
oligonucleotide is specific only for the mutant target gene.
[0083] Since recombinant retroviruses are defective, they require
assistance in order to produce infectious vector particles. This
assistance can be provided, for example, by using helper cell lines
that contain plasmids encoding all of the structural genes of the
retrovirus under the control of regulatory sequences within the
LTR. These plasmids are missing a nucleotide sequence that enables
the packaging mechanism to recognize an RNA transcript for
encapsidation. Helper cell lines that have deletions of the
packaging signal include, but are not limited to, .PSI.2, PA317 and
PA12, for example. These cell lines produce empty virions, since no
genome is packaged. If a retroviral vector is introduced into such
helper cells in which the packaging signal is intact, but the
structural genes are replaced by other genes of interest, the
vector can be packaged and vector virion can be produced.
[0084] Another targeted delivery system for antisense
polynucleotides is a colloidal dispersion system. Colloidal
dispersion systems include macromolecule complexes, nanocapsules,
microspheres, beads, and lipid-based systems including oil-in-water
emulsions, micelles, mixed micelles, and liposomes. The preferred
colloidal system of this invention is a liposome. Liposomes are
artificial membrane vesicles which are useful as delivery vehicles
in vitro and in vivo. It has been shown that large unilamellar
vesicles (LUV), which range in size from 0.2-4.0 .mu.m can
encapsulate a substantial percentage of an aqueous buffer
containing large macromolecules. RNA, DNA and intact virions can be
encapsulated within the aqueous interior and be delivered to cells
in a biologically active form (Fraley, et al., Trends Biochem.
Sci., 6:77, 1981). In addition to mammalian cells, liposomes have
been used for delivery of polynucleotides in plant, yeast and
bacterial cells. In order for a liposome to be an efficient gene
transfer vehicle, the following characteristics should be present:
(1) encapsulation of the genes encoding the antisense
polynucleotides at high efficiency while not compromising their
biological activity; (2) preferential and substantial binding to a
target cell in comparison to non-target cells; (3) delivery of the
aqueous contents of the vesicle to the target cell cytoplasm at
high efficiency; and (4) accurate and effective expression of
genetic information (Mannino, et al., Biotechniques, 6:682,
1988).
[0085] The composition of the liposome is usually a combination of
phospholipids, particularly high-phase-transition-temperature
phospholipids, usually in combination with steroids, especially
cholesterol. Other phospholipids or other lipids may also be used.
The physical characteristics of liposomes depend on pH, ionic
strength, and the presence of divalent cations.
[0086] Examples of lipids useful in liposome production include
phosphatidyl compounds, such as phosphatidylglycerol,
phosphatidylcholine, phosphatidylserine, phosphatidylethanolamine,
sphingolipids, cerebrosides, and gangliosides. Particularly useful
are diacylphosphatidylglycerols, where the lipid moiety contains
from 14-18 carbon atoms, particularly from 16-18 carbon atoms, and
is saturated. Illustrative phospholipids include egg
phosphatidyl-choline, dipalmitoylphosphatidylcholine and
distearoylphosphatidylcholine.
[0087] The targeting of liposomes can be classified based on
anatomical and mechanistic factors. Anatomical classification is
based on the level of selectivity, for example, organ-specific,
cell-specific, and organelle-specific. Mechanistic targeting can be
distinguished based upon whether it is passive or active. Passive
targeting utilizes the natural tendency of liposomes to distribute
to cells of the reticulo-endothelial system (RES) in organs which
contain sinusoidal capillaries. Active targeting, on the other
hand, involves alteration of the liposome by coupling the liposome
to a specific ligand such as a monoclonal antibody, sugar,
glycolipid, or protein, or by changing the composition or size of
the liposome in order to achieve targeting to organs and cell types
other than the naturally occurring sites of localization.
[0088] The surface of the targeted delivery system may be modified
in a variety of ways. In the case of a liposomal targeted delivery
system, lipid groups can be incorporated into the lipid bilayer of
the liposome in order to maintain the targeting ligand in stable
association with the liposomal bilayer. Various linking groups can
be used for joining the lipid chains to the targeting ligand.
[0089] 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.
[0090] For example, one of the container means may comprise a
hybridization probe that is or can be detectably labelled. A second
container may comprise a cell lysis buffer. The kit may also have
containers holding 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,
fluorescent, or radionuclide label.
[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.
EXAMPLES
[0092] The histopathologic surgical margins, lymph nodes and oral
cavity swabs from at least twenty patients were assessed for p53
mutations. Molecular analysis identified clonal tumor cells in
approximately 50% of the margins and 25% of the lymph nodes from
head and neck cancer patients initially believed to be negative by
light microscopy.
[0093] The results show that approximately 45% of invasive head and
neck squamous cell carcinoma (HNSCC) tumors have a mutated p53 gene
as determined by sequencing of primary tumor DNA.
Example 1
[0094] A. Processing 0f Histopathologic Tissue
[0095] 1. Surgical Margins
[0096] Following incision of a primary tumor, a defect is left in
the area where the tumor has been removed. Surgical margins have
been determined by frozen section and/or embedded in paraffin for
final assessment by light microscopy. In the defect that is left, a
small representative section of tissue (approximately 1
mm.times.0.5 mm) was taken from all four quadrants of each sample
and was placed in separate 1.8 ml cryostat tubes. The samples were
immediately frozen and analyzed.
[0097] 2. Lymph Node Analysis
[0098] Following standard visualization of representative sections
by light microscopy, each of the remaining lymph nodes were placed
in separate 1.8 mm cryostat tubes. The samples were immediately
frozen and analyzed.
[0099] A representative section was taken from each surgical margin
and lymph node, stained by standard H & E staining, and
visualized under light microscopy. One-hundred 12.mu. sections were
then cut and placed into 1 % SDS and 5 mg/ml of proteinase K. A
final section was take, stained by standard H & E, and also
visualized by light microscopy. The DNA was digested at 60.degree.
C. for 4 hours. The DNA was extracted twice with phenol and
chloroform by standard methods and then precipitated with ethanol.
The DNA was washed with 70% ethanol, dried, and resuspended in 50
ml of Tris-EDTA. The target p53 fragment was then amplified from
each of the surgical margins and lymph nodes as described
below.
[0100] B. PCR Amplification of Target Gene (p53)
[0101] 1. For p53 (exons 5-9), amplify 1800 bp DNA fragment:
5 Margin or lymph node DNA 5 ul Distilled water 34 ul PCR buffer
(10 X) 5 ul dNTP 3 ul Primer 4S 1 ul Primer 9AS 1 ul Taq
(polymerase, 5 U/ul) 1 ul
[0102]
6 4S 5'-GTA GGA ATT CAC TTG TGC CCT GAC TTG-3' (SEQ. ID. NO. 13)
9AS 5'-CAT CGA ATT CTG GAA ACT TTC CAC TTG AT-3' (SEQ ID NO.
14)
[0103] The sample was placed in a 500 ul tube to which 2 drops of
mineral oil were added. The sample was amplified in an Omnigene PCR
machine, as follows: 95.degree. C. for 30 sec.; 60.degree. C. for 1
min. 35 cycles; 70.degree. C. for 1 min.; and 70.degree. C. for 5
min. 1 cycle.
[0104] All amplifications were performed with negative control
(water devoid of any DNA) and positive (cell line DNA) controls,
such as SW480 (12 val mutation, K-ras) or DZ74 (273 cys mutation,
p53).
[0105] C. Cloning
[0106] To 5 ul of the PCR product above, 5ul of 2.times. stop
buffer (Bromophenol blue in Ficoll, glycerol and sarcosyl with
tris-acetate buffer) was added. The samples were run on 1 % or 2%
agarose gels to observe the yield of amplification. The remaining
45 ul of the PCR product, was mixed with 155 ul distilled water. To
the 200 ul PCR mixture, 200 ul PC-9 (Gibco-BRL), was added and
vortexed well. The tube was spun for 2 minutes in Hermle Z230 M
(Hermle, Germany) table centrifuge at high speed. The supernatant
was removed to another tube and the pellet was treated with PC-9
one more time.
[0107] The supernatant was removed to a new 1.5 ml tube and 66 ul
10 M ammonia acetate, 2 ul glycogen and 660 ul 200 proof ethanol
were added and the tube was vortexed well. The tube was spun for 20
min. in a Hermle Z230 M table centrifuge at high speed. The
supernatant was decanted and 660 ul 70% ethanol, was added and the
solution mixed. The tube was spun for 2 more minutes and decanted
again. The sample was dried in a HETOVAC (Heto, Denmark).
[0108] An alternative method for enrichment of malignant epithelial
cells and elimination of non-epithelial cells in the tissue sample,
prior to PCR, is as follows. Lymph node cells were isolated by
enzymatic digestion in 0.1 g DNAse (SIGMA) and 1 g collagenase Type
VIII (SIGMA) in 10 mm Hepes for 15-30 minutes at 37.degree. C.
Cells were washed twice in HBSS with 2% fetal calf serum (FCS) and
resuspended in 1 ml. The cell suspension was cooled on ice and 20
ug/ml of a monoclonal antibody with specificity for epithelial
cells, EBA-1 (other antibodies with specificity for epithelial
cells would be equally effective) was added to the cell suspension
and incubated on ice for one hour. The cells were washed twice in
cold (2-8.degree. C.) HBSS with 2% FCS. The cells were resuspended
in cold HBSS/2% FCS at a cell concentration of 2-4.times.10.sup.6
per ml.
[0109] Primary EBA antibodies bound to epithelial cells were
isolated on magnetic, Dynabeads (Dynal International, Oslo, Norway)
as follows. Dynabeads M-450 were coated with a secondary antibody
(sheep-antimouse, by the manufacturer). The beads were first washed
twice for 5 minutes at 2-8.degree. C. in phosphate buffered saline
(PBS), pH 7.4, containing 0.1% FCS. The Dynabeads were collected
using a magnet and the supernatant was discarded and the beads
resuspended in equivalent initial volume.
[0110] The Dynabeads coated with the secondary anti-murine antibody
at a ratio of 5 particles per target cell were added to the lymph
node sample. The concentration should be about 10.sup.7 beads per
ml of solution. The mixture was incubated for 30 minutes at
4.degree. C. on a Rock-N-Roller. Cold (4-8.degree. C.) HBSS with 1%
FCS was added in a volume at least 4.times. the volume of the
bead/cell suspension. The Dynabeads were concentrated using the
magnet. The supernatant was removed and the beads washed thoroughly
3 times using HBSS/1% FCS in a volume equal to that above (at least
4.times. the volume of the bead/cell suspension). The Dynabead/cell
suspension was centrifuged at 1,000.times.g and resuspended in
SDS/Proteinase K and DNA isolated for PCR. DNA was resuspended by
adding 4 to 8 ul of distilled water depending on the size of the
pellet. Two ul of DNA was mixed with 1 ul T4 Lambda Zap II
(Stratagene, La Jolla, Calif.) 1 ul T4 ligation buffer (5.times.).
The DNA mixture was incubated at 65.degree. C. for 5 min.,
37.degree. C. for 5 min. and 24.degree. C. for 5 min. in a water
bath. One ul of T4 ligase was added and the mixture was incubated
at 15.degree. C. for 4 to 6 hours. One ul of the ligated product
was mixed with 2.4 ul packaging extract (Stratagene) (Red) and 3.75
ul packaging extract (Yellow). The ligation mixture was kept at
room temperature for 2 hours. 250 ul phage dilution buffer (stock
phage) was added to the mixture after 2 hours.
[0111] About 10 ul to 100 ul stock phage were added to 100 ul XL1-B
cells and incubated at 37.degree. C. for 10 min. 4 ml of 55.degree.
C. top agarose was added and the mixture was plated on L-Agar gel
plates at 37.degree. C. overnight.
[0112] D. Hybridization
[0113] A piece of nylon hybridization transfer membrane (Zetaprobe,
BioRad, Richmond, Calif.) was laid on the surface of the gel which
contains lysis plaques for 1 min. The membrane was then transferred
to on a blot paper soaking 0.5 M NaOH, plaque side up for 15 min.
The membrane was then rinsed in 2.times.SSC for 5 min. twice. The
membrane was then placed on a blot paper before crosslinking under
UV light for 30 seconds. The membranes were then placed in plastic
bags for hybridization.
[0114] Oligomers (Table 2) were radioactively labeled using
.sup.32.gamma.-ATP by standard methods (T4 kinase). Sixty ng of the
oligomer (melting temperature varied from 52.degree. C. to
60.degree. C.-see Table 2) was dissolved in 6 ul distilled water. 1
ul of 10.times. kinase buffer and 2 ul of .gamma..sup.32-P-ATP were
added to the oligo and incubated at 65.degree. C for 5 minutes.
After 5 minutes the tube was spun down and 1 ul of T4 kinase was
added and incubated at 37.degree. C. for 30 minutes. The kinased
product was isolated by standard spin column protocols.
[0115] The labelled probes were added to bags containing plaque
lifts. Hybridization was performed at the temperature which is
10.degree. C. below the melting temperature of the probe for 1 hour
in a shaking bath. The membranes were then removed and washed in
3.times.SSC/0.1% SDS at room temperature for 5 min. and in
3.times.SSC/0.1% SDS at the melting temperature of the probe for 30
min. The excess solution was removed from the membrane before
wrapping in Saran Wrap. The membranes were exposed at -80.degree.
C. for 4 hours or overnight.
7TABLE 2 Oligomers Used In Head and Neck p53 Detection SEQUENCE ID
NO. CODON MT 15. 5'-CCTGCAGTAGTCCCCTG-3' (codon 126 TAC to TAG)
MT.56.degree. C. 16. 5'-CCTCAAMCAGGATGTTTTG-3' (codon 126 TAC to
TA) MT.52.degree. C. 17. 5'-GCAGCTGTGAGTTGATTC-3' (codon 146 TGG to
TGA MT.54.degree. C. 18. 5'-GCCCGGCCCCCGCGTC-3' (codon 155 ACC to
CCC) MT.62.degree. C. 19. 5'-CCGCGTCTGCGCCATG-3' (codon 158 CGC to
TGC) MT.56.degree. C. 20. 5'-GGCCATCCACAAGCAGT-3' (codon 163 TAC to
GAC) MT.54.degree. C. 21. 5'-ACAGCACACGACGGAGG-3' (codon 169 ATG to
ACG) MT.56.degree. C. 22. 5'-CGGAGGTTCTGAGGCGC-3' (codon 173 GTG to
CTG MT.56.degree. C. 23. 5'-TTGTGAGGCACTGCCCC-3' (codon 175 CGC to
CAC MT.56.degree. C. 24. 5'-AGGCGCTTCCCCCACC-3' (codon 176 TGC to
TTC MT.56.degree. C. 25. 5'-CCCCACCGTGAGCGCT-3' (codon 179 CAT to
CGT MT.56.degree. C. 26. 5'-ATCCGAGTGAAAGGAAATT-3' (codon 198 GAA
to AAA) MT.52.degree. C. 27. 5'-TGTGGAGTCTTTGGATGA-3' (codon 205
TAT to TCT) MT.52.degree. C. 28. 5'-TGTGGAGGATTTGGATGA-3' (codon
205 TAT to GAT) MT.52.degree. C. 29. 5'-TGTGGAGTGTTTGGATGA-3'
(codon 205 TAT to TGT) MT.52.degree. C. 30.
5'-ACACTTTTTGACATAGTGT-3' (codon 213 CGA to TGA) MT.50.degree. C.
31. 5-CCCCACCTTGAGCGCT-3' (codon 179 CAT to CTT) MT.54.degree. C.
32. 5'-ACATAGTATGGTGGTGCC-3' (codon 216 GTG to ATG) MT.54.degree.
C. 33. 5'-ACATAGTTTTGTGGTGCC-3' (codon 216 GTG to TTT)
MT.52.degree. C. 34. 5'-GGTGCCCTGTGAGCCG-3' (codon 220 TAT to TGT)
MT.56.degree. C. 35. 5'-GGTGCCCCATGAGCCG-3' (codon 220 TAT to CAT)
MT.56.degree. C. 36. 5'-GGTGCCCTCTGAGCCG-3' (codon 220 TAT to TCT)
MT.56.degree. C. 37. 5'-TGGCTCTGAGTGTACCAC-3' (codon 228 GAC to
GAG) MT.56.degree. C. 38. 5'-CATCCACTGCAACTACAT-3' (codon 234 TAC
to TGC) MT.52.degree. C. 39. 5'-CTACAACTAAATGTGTAACA-3' (codon 236
TAC to TAA) MT.52.degree. C. 40. 5'-CAACTACATTTGTAACAGTT-3' (codon
237 ATG to ATT) MT.52.degree. C. 41. 5'-CAACTACATATGTAACAGTT-3'
(codon 237 ATG to ATA) MT.52.degree. C. 42.
5'-ACTACATGTTTAACAGTTCC-3' (codon 238 TGT to TTT) MT.54.degree. C.
43. 5'-ACTACATGTATAACAGTTCC-3' (codon 238 TGT to TAT) MT.54.degree.
C. 44. 5'-CAGTTCCTTCATGGGCG-3' (codon 242 TGC to TTC) MT.54.degree.
C. 45. 5'-GCATGGGCGTTATGAAC-3' (codon 245 GGC to GTT) MT.52.degree.
C. 46. 5'-GCATGGGCTGCATGAAC-3' (codon 245 GGC to TGC) MT.54.degree.
C. 47. 5'-GCATGGGCGACATGAAC-3' (codon 245 GGC to GAC) MT.54.degree.
C. 48. 5'-GGCGGCTTGAACCGGAG-3' (codon 246 ATG to TTG) MT.58.degree.
C. 49. 5'-CATGAACCTGAGGCCCAT-3' (codon 248 CGG to CTG)
MT.56.degree. C. 50. 5'-GCATGAACTGGAGGCCCA-3' (codon 248 CGG to
TGG) MT.58.degree. C. 51. 5'-GCATGAACCAGAGGCCCA-3' (codon 248 CGG
to CAG) MT.58.degree. C. 52. 5'-AACCGGAGTCCCATCCTC-3' (codon 249
AGG to AGT) MT.58.degree. C. 53. 5'-GAACCGGGGGCCCATC-3' (codon 249
AGG to GGG) MT.56.degree. C. 54. 5'-GAGGCCCAACCTCACCA-3' (codon 251
ATC to AAC) MT.56.degree. C. 55. 5'-CATCACACCGGAAGACT-3' (codon 257
CTG to CCG) MT.52.degree. C. 56. 5'-TCTACTGGAACGGAACAG-3' (codon
266 GGA to GAA) MT.54.degree. C. 57. 5'-TTGAGGTGCATGTTTGTG-3'
(codon 273 CGT to CAT) MT.52.degree. C. 58.
5'-TTGAGGTGGGTGTTTGTG-3' (codon 273 CGT to GGT) MT.54.degree. C.
59. 5'-GCTGTTTATGCCTGCCT-3' (codon 275 TGT to TAT) MT.56.degree. C.
60. 5'-TGCCTGTACTGGGAGAGA-3' (codon 278 CCT to CTT) MT.56.degree.
C. 61. 5'-TGCCTGTTCTGGGAGAGA-3' (codon 278 CCT to TCT)
MT.56.degree. C. 62. 5'-TGCCTGTCGTGGGAGAGA-3' (codon 278 CCT to
CGT) MT.58.degree. C. 63. 5'-CTGTCCTGAGAGAGACC-3' (codon 279 GGG to
GAG) MT.54.degree. C. 64. 5'-CTGTCCTGGGGAGAGAC-3' (codon 279 insert
G) MT.56.degree. C. 65. 5'-TGGGAGACACCGGCGCA-3' (codon 281 GAC to
CAC) MT.58.degree. C. 66. 5'-TGGGAGAGAGCGGCGCA-3' (codon 281 GAC to
GAG) MT.58.degree. C. 67. 5'-GCGCACAAAGGAAGAGAA-3' (codon 285 GA to
AAG) MT.54.degree. C. 68. 5'-GCACAGAGAAAGAGAATCT-3' (codon 286 GAA
to AAA) MT.54.degree. C. 69. 5'-GAAAGGGTAGCCTCACC-3' (codon 294 GA
to TAG) MT.54.degree. C. 70. 5'-GAGCCTCCCCACGAGCT-3' (codon 296 CAC
to CCC) MT.58.degree. C. 71. 5'-CTCACCACTAGCTGCCG-3' (codon 298 GA
to TAG) MT.56.degree. C. 72. 5'-CTCACCACGCACTGCCC-3' (codon 298 GA
to GCA) MT.58.degree. C. 73. 5'-GGTGCGTATTTGTGCCT-3' (codon 274 GTT
to ATT MT.52.degree. C. MT = Melting temperature
[0116] Alternatively, the oligomers were labeled using
chemiluminescence. 100 pmoles of oligomers was added to 16 ul
cacodylate buffer, 10 ul fluorescein-dUDP, 16 ul terminal
transferase and water to total 160 ul. The mixture was incubated
for 1 hour at 37.degree. C.
[0117] Membranes were pre-hybridized for 1 hour in
[5.times.SSC/0.02% SDS; 0.5% (w/v) blocking agent (Milk) (Amersham,
UK)] The probe was added and hybridization was allowed to go for 1
hour at the temperature which is 10.degree. C. below the melting
temperature of the probe in shaking bath. The membranes were washed
in 3.times.SSC/0.1% SDS at room temperature 5 min. twice. The
membranes were then washed in 3.times.SSC/0.1% SDS at the melting
temperature of the probe for 15 min. three times in the washing
bath. The membranes were then rinsed in TBS for 1 minute followed
by incubation in block buffer (5% dry nonfat milk/TBS) for 30 min.
The membranes were then rinsed in TBS for 1 min. and incubated in
antibody solution (1:5000 anti-fluorescein alkaline phosphatase
antibody (Boehringer Mannheim, Indianapolis, IN) in 5% milk/TBS)
for 30 minutes. The membrane was then washed in TBS for 5 min.
eight times with shaking. The detection reagent lumigen PPD
(Boehringer Mannheim, 1:100 in MgCl.sub.2, 50 mmol/lit TBS, pH 9.5)
were mixed, and the blots incubated in the solution for 1 min. The
extra solution was removed and the membrane wrapped with plastic
and exposed to X-ray film immediately for 10 to 60 minutes.
[0118] E. Single Stranded Hybridization
[0119] Prior to the PCR reaction of the target p53 gene, either
primer must be phosphorylated at the 5' site. Half of the PCR
reaction was digested with lamda exonuclease allowing only
digestion of phosphorylated strand. The product was then run on a
spin column and DNA free of primers and enzyme was isolated. Single
stranded DNA was run on an agarose gel and transferred by standard
techniques. Oligomer specific hybridization with either
radiolabeled or chemiluminescence labeled probes was performed as
described below. A positive signal was detected by exposure to
X-ray film.
Example 2
[0120] The case for presentation was selected from a group of
individuals with HNSCC enrolled in a research protocol to
investigate p53 gene mutations in this disease. The protocol was
approved by The Johns Hopkins University Joint Committee on
Clinical Investigation. The patient had provided written informed
consent for participation in the study.
[0121] PM is a 58-year-old woman who presented to her dentist with
a history of sores in her mouth for several months. She had been a
smoker of one pack per day and had regularly used alcohol in the
past. Physical examination revealed a fungating mass in the left
retromolar trigone (RMT) which measured 5.times.3 cm. A separate,
3.times.2.5 cm ulcerative mass was noted on the right posterior
maxillary alveolar ridge (ALV). On the anterior floor of mouth
(FOM) a 5 mm patch of leukoplakia was present (FIG. 1). Biopsy of
the lesions showed that the two larger ones were both infiltrating
moderately differentiated squamous cell carcinoma (SCC) while the
FOM lesion consisted of severe dysplasia. There was a single
palpable mobile node in the left midjugular region and a second in
the right upper jugular chain, both approximately 2-cm in diameter.
A CT scan showed bony erosion of the right maxillary alveolus but
no invasion of the left mandible. Panendoscopy revealed no other
lesions of the upper aerodigestive tract.
[0122] One month after diagnosis, the patient underwent resection
of both invasive cancers via midline mandibulotomy with bilateral
neck dissections. The right palate was repaired using a temporalis
muscle flap and the left RMT was reconstructed using a pectoralis
major myocutaneous flap. Standard pathologic evaluation of the
specimens revealed all mucosal margins to be free of tumor, both on
frozen and permanent section. Deep soft tissue margins from both
the ALV and RMT resections were read as focally positive for tumor.
The right neck had one level I and one level III lymph node with
metastatic squamous carcinoma. Forty-six other lymph nodes were
negative for tumor. The left neck had a single level 11 node with
metastatic squamous carcinoma and 35 nodes with no tumor.
[0123] At surgery, samples of the three oral lesions together with
resection margins from each of the two infiltrating carcinomas were
sent for molecular analysis of the p53 genes. In addition,
formalin-fixed tissue from the lymph nodes were submitted for
analysis.
[0124] The patient went on to receive 6,600 rads external beam
radiation therapy. Six months later an ulcer developed on the right
palate posterior to the site of the resected right ALV lesion which
was biopsied and again showed SCC. A swab of the oral cavity with
gloved finger and tongue blade was obtained. The persistent tumor
was then removed by wide local excision. Once again all deep and
mucosal margins were negative for tumor on frozen section. Adjacent
marginal tissue was again sent for p53 mutation screening. The
palate defect was filled with a dental obturator. The patient
remains disease free two months after salvage surgery.
[0125] Samples from the two invasive carcinomas and the FOM
dysplastic lesion as well as the recurrent right ALV tumor were
rapidly frozen in OCT (Tissue Tek, Elkhart, Ind.), a polyglycol
embedding medium. Cryostat sections were examined with hematoxylin
and eosin stain to ensure that the tissue consisted of at least 50%
tumor cells. Over fifty 12-micron sections were then cut and placed
in SDS/proteinase K to dissolve proteins. DNA was extracted with
phenol/chloroform and precipitated with ethanol. A 1.8 kilobase
segment of the p53 gene including exons 5 to 9 was amplified by PCR
as described in Example 1 (Sidransky, D., et al., Science,
252:706-709, 1991). The UDP cloning site was added to the 5' end of
the primers to permit cloning into a clone amp (BRL) plasmid vector
(pSPORT) (See Example 3) (J. 0. Boyle et al., Cancer Research, In
press). Following amplification with uracil-containing primers, PCR
products were extracted with phenol/chloroform, gel purified, and
treated with UDP, and one half of the total product was annealed to
the plasmid vector according to the manufacturer's instructions
(Buchman, G W, et al., Focus, 14:41-45, 1992). Competent DH5-alpha
cells were transfected with plasmid by heat shock, plated onto
ampicillin plates, and incubated overnight at 37.degree. C. Over
100 colonies were pooled and DNA was isolated from plasmid by
alkaline lysis and precipitation in isopropanol.
[0126] Double-stranded DNA from plasmid was sequenced by the
dideoxy method as described in Sidransky, D., et al., supra.
Sequencing reaction products were separated on a 6%
urea/polyacrylamide gel and exposed to film. All mutations were
confirmed by repeating the PCR reaction followed by recloning and
resequencing.
Detection of Mutation DNA Sequences in Other Clinical Specimens
[0127] When specific p53 point mutations had been identified and
confirmed in the fresh tumor specimens, mutant-specific oligomer
probes were synthesized for each tumor. Probe sequences were:
8 codon 278: 5'-TGTGCCTGTACTGGGAGA-3' (RMT) (SEQ ID NO. 74) codon
281: 5'-TGGGAGACACCGGCGC-3' (ALV) (SEQ ID NO. 75)
[0128] DNA was extracted from fresh tumor margins, exfoliated cells
from oral swabs, and cervical nodal tissue as described in
Sidransky, D., et al., supra. A portion of the p53 gene (exons 7
and 8) was amplified by PCR from each sample and cloned into a
Lambda Zap phage vector (Stratagene). Phage were plated and plaques
were immobilized onto nylon filters, then probed with the two
.sup.32P labeled DNA probes specific for each missense mutation as
described in Sidransky, D., et al., supra., and in Sidransky, D.,
et al., (Science, 256:102-105, 1992)
[0129] A different missense point mutation was identified in the
p53 gene in each of the two invasive carcinomas. FIG. 2 shows an
autoradiograph of a sequence gel showing p53 mutations in DNA from
ALV tumor samples. (n)=normal DNA from peripheral blood
lymphocytes; (p)=primary ALV tumor; (r)=recurrent tumor, p and r
contain the identical G.fwdarw.C transversion (arrows) in codon
281. The left RMT trigone lesion had a C.fwdarw.A transversion in
codon 281. This mutation results in the substitution of a threonine
for proline in the p53 protein. The right ALV tumor contained a
G.fwdarw.C transversion in codon 281, resulting in an asparagine to
histidine substitution (FIG. 2). The dysplastic lesion from the
anterior floor of mouth was found to have a wild-type p53 sequence.
The recurrent right ALV tumor was found to contain the identical
281 mutation seen in the original ALV tumor six months earlier
(FIG. 2).
Evaluation of Tumor Margins
[0130] Three separate margins surrounding each tumor were obtained
at the time of the initial surgery and at resection of the
recurrent tumor. DNA from each margin was separately analyzed for
the presence of mutant sequences using the two mutant-specific
probes. All margins were free of cells containing the codon 278
mutation found in the left RMT lesion. The codon 281 probe for the
right ALV tumor identified malignant cells in one histologically
negative mucosal margin taken from the right alveolar ridge
posteromedial to the clinically detectable tumor (FIG. 3).
Evaluation of the number of phage plaques containing the mutant
sequence indicated that this margin contained approximately 28%
malignant cells (70 positive plaques of 250 total plaques, Table
2). Microscope reassessment of a parallel section from the margin
block confirmed it to be histologically negative for tumor when
reviewed after the molecular analysis.
[0131] Surgical margins from the tumor that recurred six months
later were again histologically negative for cancer cells. Samples
from three areas were analyzed for the presence of mutant p53
sequences. Mutant cells were detected in only one margin from the
posterior buccal region. Cancer cells comprised only 1 in 1,500
normal cells in this margin (Table 3).
Evaluation of Cervical Lymph Nodes
[0132] The p53 gene from two left-sided and seven right-sided
cervical lymph nodes was amplified and cloned. Two of these nodes,
one from each side, were histologically positive for tumor
involvement. Probing all nine nodes for the codon 278 mutation
demonstrated the absence of metastatic cells from the left RMT
lesion. The histologically positive node from each side contained
cells with the codon 281 mutation, indicating that the right ALV
lesion had metastasized bilaterally. Eleven percent of the plaques
from the right neck node and five percent from the left contained
the mutated segment, providing an estimate of the extent of
metastatic involvement in each node. All of the histologically
negative nodes were also negative by molecular analysis for
mutation at either codon (Table 3).
Evaluation of Exfoliated Cells in Oral Swabs
[0133] Prior to the resection of the recurrent tumor, the oral
cavity was swabbed, first with a gloved finger and then with a
tongue blade. The glove and tongue blade were then rinsed with
saline into separate containers. Probing of PCR cloned products
demonstrated the codon 281 mutation in 10% of cells from the glove
swab and in 5% from the tongue blade scraping (Table 3).
9TABLE 3 Tumor Designation Tissue Ratio Primary M.sub.1 R Posterior
(-) neg 81 GAC-CAC M.sub.2 R Post Alv. Ridge (-) 28% M.sub.3 R
Anterior (-) neg L.sub.1 R LN (+) 11% L.sub.3 L LN (+) 5% L.sub.2 R
LN (-) neg L.sub.4 L LN (-) neg R LN (-) neg L LN (-) neg Recurrent
Anterior Deep (-) neg 81 GAC-CAC Posterior Deep (-) neg Posterior
Buccal Mucosa (-) .067% Saliva (blade) 5% Saliva (glove) 10%
[0134] Molecular analysis of right (R) or left (L) alveolar ridge
(ALV) tumor, margins (M) saliva, and lymph nodes (L) or (LN).
Negative (-) and positive (+) by histopathology. Ratio denotes
percent of tumor cells by molecular analysis. Positive analysis of
saliva corresponded with recurrence of tumor.
[0135] The case presented in this example illustrates the clinical
utility of molecular analysis of p53 mutations in HNSCC, as
described in the present invention. The patient presented with
multiple mucosal lesions, including two large invasive cancers and
a small area of leukoplakia. Histologically, the two invasive
tumors looked similar. They were well demarcated and the
intervening mucosa of the hard palate appeared clinically and
histologically normal. When DNA from the three lesions was
extracted and amplified and the p53 gene was sequenced, each
invasive tumor revealed a unique mutation. The presence of
differing p53 mutations in the two tumors this case comports with
previous identification of discordant p53 mutations in multiple
primaries of HNSCC (Chung K Y, et al., Cancer Res., 53:1676-1683,
1993).
Example 3
p53 Gene Mutations
[0136] Surgically resected specimens of invasive HNSCs were
collected with consent from patients at Johns Hopkins Hospital.
Tumors were fresh frozen and later carefully
cryostat-microdissected to enrich for neo plastic cells. Cases with
less than 50% neoplastic cells were not included in the analysis.
More than fifty 12 micron sections were cut and placed in
SDS/Proteinase K followed by extraction with phenol/chloroform and
ethanol precipitation. Archival lesions consisting of dysplasia or
carcinoma in situ (CIS) were identified retrospectively (1991-1993)
through a systematic search in the files of the surgical pathology
division of the Department of Pathology. These formalin-fixed,
paraffin-embedded lesions were microdissected to enrich for
neoplastic cells and then deparaffinized in xylene. DNA from tissue
was then digested, extracted, and precipitated with ethanol as
above.
[0137] From primary fresh frozen tumor DNA, a 1.8kb segment of the
p53 gene encompassing exons 5 to 9 was amplified by the polymerase
chain reaction (PCR) as described in Example 1. A UDP cloning site
was added to the 5' end of the primers
(a=5'-CAUCAUCAUCAUTTCACTTGTGCCCTGACTT-3' (SEQ ID NO. 76) and
d=5'-CAUCAUCUACUACTGGAAACTTTCCACTTGAT-3') (SEQ ID NO. 77) to allow
cloning into a Cloneamp (BRL, Gaithersberg, Md.) plasmid vector
(pSPORT) (Buchman, G. W., et al., Focus, 14:41-45, 1992). From
archival samples the p53 gene was amplified in two segments. One
segment included exons 5 and 6 utilizing primers "a" and
b=5'-CUACUACUACUACCACTGACMCCACCCT- T-3' (SEQUENCE ID NO. 78) and
the other segment included exons 7 and 8 utilizing primers
c=5'-CAUCAUCAUCAUCCAAGGCGCACTGGCCTC-3' (SEQUENCE ID NO. 79) and
"d". Following amplification with uracil-containing primers, the
PCR products were extracted with phenol/chloroform and run on a 1%
agarose gel. The product was extracted from the gel, treated with 1
unit of uracil DNA glycosolase (UDG), and 1/2 the total product was
annealed to the plasmid vector according to the manufacturer's
instructions (Buchman, G. W. et al., supra.). Competent DH5-alpha
cells were transfected with plasmid by heat shock, plated on
ampicillin plates, and incubated overnight at 37.degree.. More than
100 colonies were pooled, and plasmid DNA was isolated by alkaline
lysis.
Sequencing
[0138] Double-stranded DNA obtained from plasmid was sequenced by
the dydeoxy method utilizing Sequenase (United States Biochemical,
Cleveland, Ohio) .sup.33P-dATP or .sup.35S-dATP (Sidransky, D., et
al., Science, 252:706-709, 1991). Prior to termination, a 30-minute
incubation with 0.5 units of Klenow fragment (USB) was added to
eliminate "stop" bands. Sequencing reaction products were then
separated on a 8M urea/6% polyacrylamide gel and exposed to film.
All mutations were confirmed by a second PCR reaction followed by
reckoning and resequencing.
Clinical Data
[0139] All information was obtained from patient records and
assessed as follows.
Tobacco Exposure
[0140] Heavy>1 pack/day
[0141] Moderate<1 pack/day or quit 5-20 years ago
[0142] Mild nonsmoker or quit>20 years ago
Alcohol Exposure
[0143] Light nondrinker, quit>20 years ago, or special occasions
only
[0144] Moderate=<12 ounces/week or quit 5-20 years ago
[0145] Heavy=>12 ounces/week
Results
[0146] To determine the relative timing of p53 gene mutations in
HNSCC, 65 fresh primary invasive tumor samples and 37 archival
preinvasive lesions were sequenced. Nineteen percent ({fraction
(7/37)}) of the early preinvasive lesions contained p53 mutations,
compared to 43% ({fraction (28/65)}) of the primary invasive HNCS,
(Table 4). Only seven p53 mutations were found in preinvasive
lesions: {fraction (5/24)} in CIS lesions (21 %) and {fraction
(2/13)} in dysplastic lesions (15%). Both of the latter mutations
were in lesions of severe dysplasia; no lesions of mild or moderate
dysplasia contained p53 mutations. The difference in incidence of
p53 mutations between noninvasive and invasive lesions was found to
be statistically significant (p <0.02 by chi-squared
analysis).
[0147] Closer analysis of the specific p53 mutations found in all
lesions reveals that 72% ({fraction (26/36)}) were missense
mutations and 28% ({fraction (10/36)}) would produce truncated
proteins. This represents a high percentage of mutations resulting
in truncations, similar to that seen in esophageal cancer
(Hollstein, M., et al., 253:49-53, 1991). Of these, four were
nonsense mutations, five were frame shift mutations and one was an
altered splice site mutation.
[0148] Interestingly, one of these mutations (Hi8) was a deletion
that occurred near small repeating sequences, perhaps secondary to
replication errors as described in Jejo, N., et al., (Oncogene,
8:209-213, 1993). Additionally, one tumor (H15) contained an
unusual pyrimidine dimer 2 bp mutation at codon 245. Although
common in UV-induced skin tumors similar mutations have been
previously seen in bladder cancer and may also be induced by
reactive oxygen free radicals or severe exposure to carcinogens
(Spruck, C. H., et al., Can. Res., 53:1162-1166, 1993). Eighty-two
percent of the invasive tumors ({fraction (23/28)}) had lost the
wild type allele, suggesting loss of the remaining p53 allele
during tumor progression. One tumor (H10) contained two point
mutations, and analysis of individual clones revealed that each
occurred on a different allele. In contrast, {fraction (5/7)} p53
mutations in early lesions appeared to have retained of the wild
type allele. The presence of this allele may represent some
residual contamination from surrounding non-neoplastic tissue, or
it may mean that the second allele had not yet been lost in these
early lesions. Mutations were spread over a wide range of codons
yet occurred in the most highly conserved regions of p53 (FIG. 4A
and 4B) as previously reported (Hollstein, M., et al., supra.). The
mutations also occupied a spectrum similar to that seen in SC
carcinoma of the lung (Y. Kishimoto et al., Can. Res. 52:4799-4804,
1992), but they differed from mutations seen in other epithelial
tumors.
[0149] Clinical data were available for 26 of the 27 patients with
primary invasive carcinomas containing mutations in the p53 gene
(Table 4). Of these, {fraction (16/25)} point mutations (64%) were
at guanine nucleotides, including 8 G.fwdarw.A, 5G.fwdarw.T, or
3G.fwdarw.C changes often associated with benzopyrenes,
nitrosamines, and possibly oxygen radicals from cigarette smoke
(Kishimoto, Y., et al., Can. Res., 52:4799-4804, 1992; Puisieux,
A., et al., Can. Res., 51:6185-6189, 1991; Ames, B. N., Science,
221:1256-1264, 1983; Kalra, J., et al., Int J. Exp. Pathol.,
72:1-7, 1991). Thirteen of these 16 patients were heavy smokers,
and five of them also had histories of heavy drinking. Only one
patient with a G.fwdarw.A transition was a nonsmoker and
nondrinker. Overall, 25 of 26 patients with mutations had histories
of moderate to heavy smoking. In contrast, eleven of 38 patients
without p53 mutations had had only minimal exposure to tobacco and
alcohol. Although the exact amount was quite variable and difficult
to quantify accurately, carcinogen exposure was significant in all
but one of the patients with missense mutations. Exposure was
particularly heavy in the group with specific mutations previously
found to be associated with known carcinogens in cigarette smoke.
The presence or absence of p53 mutations did not correlate with age
or sex of the patient nor with the site, size, or stage of the
tumor.
[0150] One patient (H27) had two separate tumors on opposite sides
of the oral cavity (Table 4). Each tumor had a distinct p53
mutation as previously seen in patients with separate primaries
(Chung, K. Y., et al., Can. Res., 53:1676-1603, 1993). One lesion
from another patient (C7) with a G.fwdarw.A transition was a
nonsmoker and nondrinker. Overall, 25 of 26 patients with mutations
had histories of moderate to heavy smoking. In contrast, eleven of
38 patients without p53 mutations had had only minimal exposure to
tobacco and alcohol. Although the exact amount was quite variable
and difficult to quantify accurately, carcinogen exposure was
significant in all but one of the patients with missense mutations.
Exposure was particularly heavy in the group with specific
mutations previously found to be associated with known carcinogens
in cigarette smoke. The presence or absence of p53 mutations did
not correlate with age or sex of the patients nor with the site,
size, or stage of the tumor. Clinical data were not available for
patients with noninvasive (dysplastic or CIS) lesions.
[0151] As shown in FIG. 5, only the carcinomatous portion of the
neoplasm stained with an anti-p53 antibody (indicative of p53
mutation) and the ABC VectaStain.RTM. kit (Vector Laboratories)
(Cunningham, J., et al., Can. Res., 52:1974-1980, 1992).
Microdissection of the distinct histologic regions followed by DNA
extraction and p53 sequencing revealed a codon 175 mutation in the
carcinoma, while no mutations were detected in the papilloma. This
case was stained simply to illustrate the histopathologic
progression to cancer associated with a new p53 mutations.
[0152] Table 5 lists the known p53 mutations associated with head
and neck tumors.
10TABLE 4A PRIMARY INVASIVE HNSC TUMORS WITH P53 MUTATIONS STAGE
TOB/ETOH SITE AGE SEX CODON MUTATION AMINO ACID H1 T2N0 H/M L 41 M
275 TGT.fwdarw.TAT CYS.fwdarw.TYR H2 T3N0 H/L L 67 F 296
CAC.fwdarw.CCC HIS.fwdarw.PRO H3 T3N0 M/M L 80 M 163 TAC.fwdarw.CAC
TYR.fwdarw.ASP H4 T3N25 H/H L 65 M 175 CGC.fwdarw.CAC
ARG.fwdarw.HIS H5 T2N2 H/H OP 36 M 296 GAG.fwdarw.TAG
GLU.fwdarw.TERM H6 T2N0 H/H OP 55 F 234 TAC.fwdarw.TCC
TYR.fwdarw.CYS H7 T4N2C H/H OP 55 M 278 T insertion FS H8 T3N0 H/L
L 58 M 228 GAC.fwdarw.GAG ASP.fwdarw.GLU H9 N/A N/A L N/A N/A 296
GAG.fwdarw.TAG GLU.fwdarw.TERM H10 T1N0 H/L HP 59 M 175
CGC.fwdarw.CAC ARG.fwdarw.HIS 216 GTG.fwdarw.ATC VAL.fwdarw.MET H11
T2N1 H/H OC 68 M * acceptor G.fwdarw.T SPLICE SITE H12 T3N1 M/H OC
84 M 220 TAT.fwdarw.TCT TYR.fwdarw.SER H13 T3N1 H/L L 64 M 198
GAA.fwdarw.AAA GLU.fwdarw.LYS H14 T1N0 M/H OC 65 M 205
TAT.fwdarw.GAT TYR.fwdarw.ASP H15 T1N1 M/H OC 68 F 245
GGC.fwdarw.GTT GLY.fwdarw.VAL H16 T2N0 H/L L 68 F 257
CTG.fwdarw.CCG LEU.fwdarw.PRO H17 T2N2A H/H L 46 M 220
TAT.fwdarw.TGT TYR.fwdarw.CYS H18 T2N0 H/H OC 64 F 257-61 9bp
deletionFS H19 T3N0 H/H OP 70 F 278 CCT.fwdarw.TCT PRO.fwdarw.SER
H20 T2NC H/M OP 46 M 220 TAT.fwdarw.TGT TYR.fwdarw.CYS H21 T4N2C
H/M L 47 M 279 G insertion FS H22 T4N0 L/L OC 85 F 248
CCG.fwdarw.CAG ARG.fwdarw.GLN H23 TxN2 H/M NE 61 M 237
ATG.fwdarw.ATT MET.fwdarw.ILE H24 T3N2C H/H L 58 M 251
ATC.fwdarw.AAC ILE.fwdarw.ASN H25 T2N2C H/NA L 66 M 230
TAC.fwdarw.TAA TYR.fwdarw.TERM H26 T2NO H/L OC 50 F 273
CGT.fwdarw.GGT ARG.fwdarw.GLY H27A T3N0 H/H OC 58 F 278
CCT.fwdarw.ACT PRO.fwdarw.THR 14N2C H/H OC 58 F 281 GAC.fwdarw.CAC
ASP.fwdarw.HIS Tob = Tobacco; ETOH = Ethanol; L = Light; M =
Moderate; H = Heavy; N/A = Not Available FS = Frame Shift; L =
Larynx; OP = Oropharynx; OR = Oral Cavity; NE = Neck *Intron 5
(-3bp from exon 6). For assessment of tobacco and ETOH exposure see
Example 3 text.
[0153]
11TABLE 4B NONINVASIVE TUMORS PATHOLOGY SITE CODON MUTATION AMINO
ACID C1 CIS OC 200 A deletion FS C2 CIS OC 148 TGG.fwdarw.TGA
TRP.fwdarw.TERM C3 dys OC 249 A insertion FS C4 dys OC 169
ATG.fwdarw.ACG MET.fwdarw.THR C5 CIS OC 278 CCT.fwdarw.ACT
PRO.fwdarw.THR C6 CIS OC 168 CGC.fwdarw.TGC ARG.fwdarw.CYS C7 CIS
OC 176 TGC.fwdarw.TTC CYS.fwdarw.PHE Tob = Tobacco; ETOH = Ethanol;
L = Light; M = Moderate; H = Heavy; N/A = Not Available FS = Frame
Shift; L = Larynx; OP = Oropharynx; OC = Oral Cavity; NE = Neck
*Intron 5 (-3 bp from exon 6). For assessment of tobacco and ETOH
exposure see Example 3 text.
[0154]
12TABLE 5 P53 MUTATIONS IN HEAD AND NECK TUMORS Codon Base pair
change 126 tac-tag 132 aag-agg 146 tgg-tga 148-151 10 bp deletion
FS 154 1 bp deletion FS 155 acc-ccc 158 cgc-tgc 163 tac-cac 169
atg-acg 173 gtg-ctg 175 cgc-cac 176 tgc-ttc 176 tgc-ttc 179 cat-cgt
198 gaa-aaa 200 a deletion FS 203 1 bp deletion FS 205 tat-gat 205
tat-tct 205 tat-tgt 205 tat-tgt 209 2 bp deletion FS 213 cga-tga
213 cga-cgg 213 cga-tga 216 gtg-atg 216 gtg-ttt 220 tat-tgt 220
tat-cat 220 tat-tct 228 gac-gag 234 tac-tgc 236 tac-taa 237 atg-att
237 atg-ata 238 tgt-tat 238 tgt-ttt 242 tgc-ttc 245 ggc-gtt 245
ggc-gac 245 ggc-tgc 246 atg-ttg 248 cgg-tgg 248 cgg-ctg 248 cgg-tgg
248 cgg-tgg 248 cgg-cag 248 cgg-ctg 249 agg-ggg 249 agg-agt 249 a
insertion FS 251 atc-aac 257 ctg-ccg 257-61 9 bp deletion 266
gga-gaa 271 1 bp deletion FS 273 cgt-ggt 273 cgt-cat 275 tgt-tat
278 t insertion FS 278 cct-tct 278 cct-act 278 cct-cgt 278 cct-act
279 g insertion 279 ggg-gag 281 gac-cac 281 gac-gag 281 gac-cac 285
gag-aag 286 gaa-aaa 289-290 2 bp (tt) addition FS 294 gag-tag 296
cac-ccc 298 gag-gca 298 gag-tag 298 gag-tag 305-306 taag insertion
307 16 bp deletion FS Intron 5 acceptor g-t FS = Frame Shift
Example 4
Additional Case Studies
[0155] Invasive head and neck squamous carcinomas were surgically
resected at Johns Hopkins Hospital and portions of the neoplasms
were collected with the consent of the patient. After the primary
tumor was removed and the margins were sampled by frozen section to
confirm the adequacy of resection, additional normal appearing
tissue was taken from the edges of the surgical defect. Also,
portions of lymph nodes from neck dissection specimens that were
not used for diagnostic histopathology were fresh frozen. The DNA
was prepared from al tissues as described in Example 1 and by
Sidransky, et al., (Science, 252:706, 1991).
[0156] A. Methods
[0157] 1. Histopathologic Exam
[0158] Portions of the primary carcinomas, surgical margins, and
lymph nodes were processed and sectioned in an identical manner to
guarantee accurate histopathologic assessment before molecular
analysis was performed. The frozen specimens were embedded in OCT
(Tissue Tek, Elkhart, Ind.), a polyglycol embedding medium, and the
frozen specimen block was evenly planed with a cryostat, resulting
in a smooth surface for sectioning. First, two 5-micron thick
sections were taken for hematoxylin and eosin (H&E) staining
and examination by light microscopy. The slides were read as
negative, suspicious, or positive for the presence of squamous
carcinoma. Next, twenty 12-micron sections were cut and placed in
SDS/Proteinase K for DNA analysis. The tissue DNA was extracted
with phenol/chloroform and precipitated with ethanol as described
(Boyle, J, Hakim J, Koch W, et al., Can. Res., 53:4477-80, 1993). A
second set of two H&E sections was taken, followed by a second
set of 12-micron sections for DNA analysis, and then a third set of
H&E sections. Two hundred and forty microns of tissue for DNA
analysis was, therefore, immediately sandwiched between sections
examined by light microscopy.
[0159] 2. p53 Gene Sequencing
[0160] A 1.8 kb fragment of the p53 gene encompassing exons five to
nine was amplified from the fresh-frozen primary tumor DNA by the
polymerase chain reaction (PCR) as described in Example 1 and
Sidransky, et al., 1991, supra and Boyle J, et al., supra. A UDP
cloning site was added to the 5' end of the primers and the DNA was
cloned into a CloneAmp (Gibco/BRL) plasmid vector. Competent
DH5-alpha cells were then transfected with plasmids using the
heatshock method (Buchman G W., et al., Focus, 14:41-45, 1992). The
transformed cells were plated on ampicillin plates and incubated at
37.degree. C. overnight. Colonies were pooled and plasmid DNA was
extracted by alkaline lysis. The double-stranded DNA was sequenced
by the dideoxy method, using .sup.32P-dATP (New England Nuclear),
Sequenase (USB), and Klenow enzyme (NEB) (Boyle, et al., supra).
The sequencing reactions were then electrophoreses on 8M urea/ 6%
polyacrylamide gels, fixed, and exposed overnight to film at room
temperature.
[0161] 3. Molecular Probing
[0162] The patients found to have p53 mutations in their primary
HNSC were selected for further analysis. DNA extracted from the
sectioned margins and lymph nodes was used to amplify exons five to
nine of the p53 gene by PCR as described (Sidransky, D., et al.,
Science, 252:706-709, 1991; Sidransky, D., Nature, 355:846-847,
1992). The PCR products were then cloned into a bacteriophage
vector and amplified further in Escherichia coli (Sidransky, D., et
al., Science, 252:706-709, 1991). Between 500 and 10,000 clones
were then transferred to nylon membranes and hybridized to
.sup.32P-end labeled oligonucleotide probes. These oligonucleotide
probes were unique and specific for the mutant p53 base pair found
in each patient's respective primary HNSC. Following hybridization,
the membranes were washed stringently at 54-60.degree. C. to detect
only mutant-specific binding. The membranes were then exposed to
film and positive-hybridizing plaques identified the presence of a
p53 mutant gene. The percentage of clonal (mutated) tumor cells in
each specimen was estimated by counting the number of labeled
plaques and dividing this number by the total number of plaques
present on each plate that contained the inserted p53 DNA fragment
(all plaques that hybridized to a wild-type p53 probe).
[0163] The assay was confirmed using positive and negative controls
for each margin and lymph node examined. The positive control was
the amplified p53 gene product derived from each respective primary
carcinoma; it was detected by hybridization to its mutant-specific
oligonucleotide prove. The negative control included "cloned" PCR
products from reactions devoid of DNA and cloned p53 products
derived from patients with a different p53 mutation in the primary
tumor. All positive assays were repeated by reamplification,
recloning, and reprobing.
[0164] B. Results
[0165] 1. Study Population
[0166] Forty seven patients with invasive HNSC who were scheduled
for tumor resection at Johns Hopkins Hospital entered the study.
Following sequencing of the primary tumor DNA, 21 patients (45%)
were found to have p53 gene mutations in their neoplasms (Table 6).
The 21 patients with p53 mutations consisted of 11 females and 10
males with an average age of 62 years (range was 46 years to 85
years). Twenty of 21 patients had significant tobacco use and 16 of
the 21 patients had a history of heavy alcohol consumption. The
patients most commonly presented with advanced-stage or recurrent
squamous carcinoma of the head and neck.
[0167] Sixteen of the 21 patients with p53 mutations had surgical
margins, four had both surgical margins and lymph nodes, and one
had only lymph node tissue available for further molecular
analysis. A total of 56 margins from 20 patients (average of 2.8
margins/patient) and 27 cervical lymph nodes from five patients
(average of 5.4 nodes/patient) were studied. Five patients had
positive surgical margins in the operating room on final permanent
histopathologic diagnosis and were excluded from further analysis.
Fifteen of the 20 patients with surgical margins available for
molecular analysis were found to have negative resection margins
without evidence of microscopic carcinoma documented on the final
pathology report from their HNSC operations (FIG. 6).
[0168] 2. Surgical Margins
[0169] The "negative" surgical margins from the 15 patients
described above were probed with the specific p53 mutant
oligonucleotide derived from their primary tumors (Table 7). Nine
of the 15 patients (60%) had at least one surgical margin that
specifically hybridized to the mutant probe, thereby demonstrating
the presence of mutation-containing neoplastic cells (FIGS. 7A-7C).
FIG. 7 shows a molecular anlaysis of surgical margins and lymph
nodes. Autoradiographs of plaque lifts hybridized with
mutant-specific oligomers derived from each patient's tumor are
shown. Positive (specific) hybridizing clones (black dots) are
detected in surgical margins (M), in lymph nodes (L), and in the
primary tumor (T) as a positive control.
[0170] FIG. 7A (patient 4), shows that tumor cells are identified
in one margin (M) and lymph nodes (L1, L4, and L6) with negative
hybridization (empty circles) in L2 and L3.
[0171] FIG. 7B (patient 9), shows that many tumor cells are present
in M4 and M5 and fewer tumor cells in M1 and M2, with margin M3
free of tumor cells.
[0172] FIG. 7C (patient 16), shows that micrometastases are seen in
all 4 lymph nodes (L1-L4) to varying degrees. Details of each
patient in FIGS. 7A, B, and C, and percentage of tumor cells in
margins and lymph nodes appear in Tables 7, 8, and 9.
[0173] Review of the fresh-frozen surgical margins sent for
molecular analysis revealed microscopic cancer in three of these
nine patients (FIG. 6). Those surgical margins that were positive
for squamous carcinoma based on light microscopy consistently
demonstrated significant mutant-specific hybridization on molecular
analysis (Table 7). The percentage of cells with mutations in the
surgical margins ranged from 0.05% to 10.0% by molecular analysis.
The positive margins diagnosed by histopathology consistently had
at least 5.0% neoplastic cells by molecular probing. Conversely,
when the percentage of cells harboring p53 mutations comprised less
than 5.0% of the cell population in the surgical margins, they were
not definitively detected by light microscopic examination of the
H&E sections. The remaining six patients had margins that did
not hybridize to the mutant-specific probes, suggesting that those
surgical margins did not harbor neoplastic cells (FIG. 6).
Representative sections from positive, suspicious, and negative
margins by histopathologic assessment are shown in FIGS. 8A, 8B,
and 8C, respectively. All three cases illustrated were positive by
molecular analysis. FIG. 8 shown a photomicrographs of
histopathologic margins. Hematoxylin and eosin staining of positive
FIG. (8A), suspicious FIG. (8B), and negative FIG. (8C) surgical
margins are demonstrated. All of the above margins had tumor cells
detected by molecular analysis. The percentage of neoplastic cells
was 10% in 8A (M2 from patient 13), 5% in 8B (M4 from patient 9),
and 0.25% in 8C (M2 from patient 15).
[0174] 3. Cervical Lymph Nodes
[0175] Sandwich sections of 27 cervical lymph nodes from five
patients with HNSC were also carefully examined before molecular
analyses were performed. Three of the five patients were diagnosed
with one cervical lymph node positive for the presence of
metastatic carcinoma by light microscopy (Table 8). Thus, three of
the 27 lymph nodes had evidence of metastatic HNSC on standard
histopathologic examination. In contrast, when the cloned DNA from
the lymph nodes was probed with the respective p53 mutant-specific
oligonucleotide derived from their primary tumor, positive
hybridization was identified in nine of the 27 nodes from four of
the 5 patients. Therefore, of the 24 lymph nodes negative by light
microscopy, six (25%) were found by molecular analysis to contain
neoplastic cells. Neoplastic cells comprised between 0.3% and 10.0%
of the total cell population in these positive nodes (FIG. 7). As
was true for the margins, the lymph nodes diagnosed as positive for
HNSC by light microscopy all contained a population of at least
5.0% mutant cells (Table 8). Three of the four patients with occult
metastases identified by probing would have had their head and neck
cancers upgraded to a more advanced stage based on the molecular
analysis.
[0176] Primary tumor from all 21 patients showed positive
hybridization on southern blot analysis of p53 amplified PCR
products derived from tumor DNA using their individually
synthesized oligonucleotide probe (Sidransky, D., et al., Science,
256:102-105, 1992). The patients' samples also consistently
demonstrated negative hybridization with an oligonucleotide probe
derived from the sequence of a different p53 mutation.
[0177] 4. Patient Survival
[0178] After a brief follow-up period (mean 6.4 months), 18 of the
21 patients studied were alive and three had succumbed to their
cancer. Twelve of the surviving patients are currently
disease-free, five have locoregional tumor recurrence, and one has
distant metastatic disease. It is noteworthy that the location of
the tumor margins positive by molecular analysis accurately
predicted the site of local recurrence in two patients.
13TABLE 6 CHARACTERISTICS OF PATIENTS WITH HNSC Patient # Age/Sex
Site Stage p53 Mutation 1 66/F Larynx T3NoMo 257 CTG-CCG 2 59/M
Larynx T1NoMo(R) 175 CGC-CAC 3 66/F opx T3N1Mo(R) 245 CCG-CTT 4
46/M Hypopx T2N2aMo 220 TAT-TGT 5 49/M opx T3NoMo 275 TGT-TAT 6
54/F opx T2NoMo 257 9BP Del 7 66/M opx T3N2aMo 187 GGT-GAT 8 51/M
Hypopx T4N1Mo 193 CAT-CGT 9 63/M opx T3N2bMo 306 CGA-TGA 10 35/F oc
T4NoMo 248 CGG-GGG 11 56/M Hypopx T4N1Mo 255 ATC-TTC 12 62/F opx
T1N1Mo 278 CCT-CGT 13 56/F Hypopx T4N2Mo 298 GAG-TAG 14 57/M Larynx
T2NoMo(R) 228 GAC-GAG 15 59/M opx T4N2bMo 220 TAT-TGT 16 65/M
Hypopx T3N2bMo 175 CGC-CAC 17 68/F Larynx T1NoMo(R) 272 GTG-GAG 18
67/M Larynx T3NoMo 253 ACC-TCC 254 ATC-TTC 19 73/F Larynx T2NoMo(R)
180 GAG-TAG 20 72/F oc T1NoMo(R) 249 AGG-GGG 21 62/F opx T4N3Mo 213
CGA-TGA opx = Oropharyngeal Hypopx = Hypopharynx oc = Oral Cavity
(R) = Recurrent Tumor
[0179]
14TABLE 7 MOLECULAR ANALYSIS OF SURGICAL MARGINS Surgical
Histopath. Mutant-specific Mutant Clones Patient # Margins Exam
Probing (%) 1 M1 Neg. Pos. 0.35% M2 Neg. Pos. 0.5% M3 Neg. Neg. --
M4 Neg. Pos. 0.1% M5 Neg. Pos. 0.2% M6 Neg. Pos. 0.5% 2 M1 Neg.
Pos. 0.1% M2 Neg. Pos. 0.25% M3 Neg. Pos. 0.05% M4 Neg. Pos. 0.2% 3
M1-M2 Neg. Neg. -- 4 M1 Neg. Pos. 5.0% 5 M1-M3 Neg. Neg. -- 6 M1-M3
Neg. Neg. -- 7 M1-M2 Neg. Neg. -- 8 M1 Neg. Neg. -- M2 Neg. Pos.
4.0% 9 M1 Neg. Pos. 0.2% M2 Neg. Pos. 0.5% M3 Neg. Neg. -- M4
Suspic. Pos. 5.0% M5 Pos. Pos. 10.0% 10. M1 Neg. Pos. 0.4% M2 Neg.
Pos. 1.3% 11 M1-M2 Neg. Neg. -- M3 Suspic. Pos. 0.2% M4 Suspic.
Pos. 0.7% 12 M1 Neg. Neg. -- M2 Neg. Neg. -- 13 M1 Neg. Neg. -- M2
Pos. Pos. 10.0% 14 M1-M8 Neg. Neg. -- 15 M1 Neg. Neg. -- M2 Neg.
Pos. 0.25% Each consecutive letter indicates a separate surgical
margin. Histopathologic exam was performed by staff surgical
histopathologists, and the slides were read as positive ("Pos."),
suspicious ("Suspic."), or negative "Neg.") for squamous cell
carcinoma. Forty percent mutant clones is equal to number of
mutant-specific clones/total clones with p53 insert (see text).
[0180]
15TABLE 8 MOLECULAR ANALYSIS OF CERVICAL LYMPH NODES Cervical
Histopath. Molecular Percent Mutant Patient # Nodes Exam Probing
Clones 1 L1 Positive Positive 10.0% 2 L1-L10 Negative Negative --
L11 Negative Positive 0.35% 3 L1-L3 Negative Negative -- 4 L1
Positive Positive 10.0% L2-L3 Negative Negative -- L4 Negative
Positive 1.0% L5 Negative Negative -- L6 Negative Positive 2.0%
L7-L8 Negative Negative -- 16 L1 Positive Positive 5.0% L2 Negative
Positive 0.4% L3 Negative Positive 0.3% L4 Negative Positive 0.7%
Each consecutive letter represents a separate cervical lymph node.
The slides were read as positive, suspicious, or negative for
squamous cell carcinoma.
[0181] The results shown in these examples provide an embodiment
wherein successful detection of neoplasia was accomplished and
provides a practical basis for a new approach for detecting the
presence of neoplasias, such as in histologic margins and regional
lymph nodes. The approach would have utility in monitoring patient
populations and treatments designed to minimize the incidence of
neoplasia. It also could be used in screening asymptomatic
patients, especially those at risk by virtue of inherited or
environmental factors, such as tobacco and alcohol consumption, for
the presence of neoplasia. The current results indicate that a
significant fraction of metastases and dangerous pre-malignant
lesions can be identified through this strategy. Additionally,
these findings indicate that other mutant nucleotide sequences,
besides p53, which are associated or indicative of neoplasias, such
as mutant proto oncogenes and tumor suppressor genes, would also be
detectable.
[0182] A principal advantage of the method of this invention is
that the PCR method of amplification can be used to detect one
cancer cell in ten thousand cells in tissue margins of tumors. This
high sensitivity is far superior to results obtained by the prior
art methods of histologic examination. Moreover, this invention
provides the diagnostician with an opportunity to screen tissue
from patients determined to be at risk of developing tumors at an
early stage before discernible tumors actually develop so that gene
therapy and or antisense therapy for such patients can commence at
the time most likely to result in a successful outcome.
[0183] Although the invention has been described with reference to
the presently preferred embodiment, it should be understood that
various modifications can be made without departing from the spirit
of the invention.
Sequence ID Listing
[0184] SEQUENCE ID NO. 1-2 are nucleotide sequences to which
oligonucleotide primers for sequences flanking p53 mutations in
exons 5-6, hybridize.
[0185] SEQUENCE ID NO. 3-4 are nucleotide sequences to which
oligonucleotide primers for sequences flanking p53 mutations in
exons 7-8, hybridize.
[0186] SEQUENCE ID NO. 5-8 are nucleotide sequences for primers
which hybridize to flanking sequences of p53 mutations.
[0187] SEQUENCE ID NO. 9-11 are nucleotide sequences for mutant
nucleotides in p53 at codons 273, 278, and 281, respectively.
[0188] SEQUENCE ID NO. 12 is the nucleotide sequence for a portion
of the wild type p53.
[0189] SEQUENCE ID NO. 13 is the nucleotide sequence for a primer
4S for amplification of exons 5-9 of p53.
[0190] SEQUENCE ID NO. 14 is the nucleotide sequence for a primer
9AS for amplification of exons 5-9 of p53.
[0191] SEQUENCE ID NO. 15-73 are the nucleotide sequences for
oligomers which are used to identify mutations in p53 which are
associated with head and neck tumors.
[0192] SEQUENCE ID NO. 74-75 are the nucleotide sequences for
mutant specific probes for codons 278 and 281 of p53,
respectively.
[0193] SEQUENCE ID NO. 76-79 are the nucleotide sequences for
primers used to amplify a 1.8 kb segment of the p53 gene
encompassing exons 5-8.
[0194] SEQUENCE ID NO. 80-82 are the nucleotide sequences for
probes for detection of p53 mutations.
Sequence CWU 1
1
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