|
Cancer Patent Abstract
The present invention is directed to a method for determining the
responsiveness of cancer to an epidermal growth factor receptor
(EGFR) treatment. In a preferred embodiment, the presence of at
least one variance in the kinase domain of the erbB1 gene confers
sensitivity to the tyrosine kinase inhibitor gefitinib. Thus, a
diagnostic assay for these mutations will allow for the administration
of gefitinib, erlotinib and other tyrosine kinase inhibitors to
those patients most likely to respond to the drug.
Cancer Patent Claims
We claim:
1. A method for determining an increased likelihood of pharmacological
effectiveness of treatment by gefitinib or erlotinib in an individual
diagnosed with non-small cell lung cancer comprising: obtaining
DNA from a non-small cell lung cancer tumor sample from the individual;
and determining the presence or absence of at least one nucleotide
variance in exon 18, 19, or 21 of the epidermal growth factor receptor
(EGFR) gene in the DNA, wherein the presence of at least one nucleotide
variance selected from: 1) an in-frame deletion in exon 19 of the
EGFR gene consisting of a deletion within codons 746 to 753 that
results in amino acid changes comprising a deletion of at least
amino acids leucine, arginine, and glutamic acid at position 747,
748, and 749 of SEQ ID NO:512; 2) a substitution in exon 21 that
results in an amino acid change consisting of a substitution of
arginine for leucine at position 858 (L858R) of SEQ ID NO:512, or
a substitution in exon 21 that results in an amino acid change consisting
of a substitution of glutamine for leucine at position 861 (L861Q)
of SEQ ID NO:512; or 3) a substitution in exon 18 that results in
an amino acid change consisting of a substitution of cysteine for
glycine at position 719 (G719C) of SEQ ID NO:512 indicates an increased
likelihood of pharmacological effectiveness of treatment by gefitinib
or erlotinib in the individual.
2. The method of claim 1, wherein the presence or absence of the
at least one nucleotide variance is determined by DNA sequencing.
3. The method of claim 1, wherein the presence or absence of the
at least one nucleotide variance is determined by allele-specific
amplification.
4. The method of claim 1, wherein the presence or absence of the
at least one nucleotide variance is determined by single strand
conformation polymorphism, denaturing gradient gel electrophoresis
or temperature gradient gel electrophoresis analysis.
5. The method of claim 1, wherein the presence or absence of the
at least one nucleotide variance is determined by mismatch cleavage
analysis.
6. The method of claim 1 wherein the at least one nucleotide variance
is an in-frame deletion in exon 19 of the EGFR gene consisting of
a deletion within codons 746 to 753 that results in amino acid changes
comprising a deletion of at least amino acids leucine, arginine,
and glutamic acid at position 747, 748, and 749 of SEQ ID NO:512.
7. The method of claim 1, wherein the at least one nucleotide variance
is a substitution in exon 21 that results in an amino acid change
consisting of a substitution of arginine for leucine at position
858 (L858R) of SEQ ID NO:512 or a substitution of glutamine for
leucine at position 861 (L861Q) of SEQ ID NO:512.
8. The method of claim 1, wherein the at least one nucleotide variance
is a substitution in exon 18 that results in an amino acid change
consisting of a substitution of cysteine for glycine at position
719 (G719C) of SEQ ID NO:512.
Cancer Patent Description
SEQUENCE LISTING
The instant application contains a "lengthy" Sequence
Listing which has been submitted via CD-R in lieu of a printed paper
copy, and is hereby incorporated by reference in its entirety. Said
CD-R, recorded on Nov. 25, 2005, are labeled CRF, "Copy 1"
and "Copy 2", respectively, and each contains only one
identical 445 KB file (055147.APP).
BACKGROUND
Epithelial cell cancers, for example, prostate cancer, breast cancer,
colon cancer, lung cancer, pancreatic cancer, ovarian cancer, cancer
of the spleen, testicular cancer, cancer of the thymus, etc., are
diseases characterized by abnormal, accelerated growth of epithelial
cells. This accelerated growth initially causes a tumor to form.
Eventually, metastasis to different organ sites can also occur.
Although progress has been made in the diagnosis and treatment of
various cancers, these diseases still result in significant mortality.
Lung cancer remains the leading cause of cancer death in industrialized
countries. Cancers that begin in the lungs are divided into two
major types, non-small cell lung cancer and small cell lung cancer,
depending on how the cells appear under a microscope. Non-small
cell lung cancer (squamous cell carcinoma, adenocarcinoma, and large
cell carcinoma) generally spreads to other organs more slowly than
does small cell lung cancer. About 75 percent of lung cancer cases
are categorized as non-small cell lung cancer (e.g., adenocarcinomas),
and the other 25 percent are small cell lung cancer. Non-small cell
lung cancer (NSCLC) is the leading cause of cancer deaths in the
United States, Japan and Western Europe. For patients with advanced
disease, chemotherapy provides a modest benefit in survival, but
at the cost of significant toxicity, underscoring the need for therapeutic
agents that are specifically targeted to the critical genetic lesions
that direct tumor growth (Schiller J H et al., N Engl J Med, 346:
92-98, 2002).
Epidermal growth factor receptor (EGFR) is a 170 kilodalton (kDa)
membrane-bound protein expressed on the surface of epithelial cells.
EGFR is a member of the growth factor receptor family of protein
tyrosine kinases, a class of cell cycle regulatory molecules. (W.
J. Gullick et al., 1986, Cancer Res., 46:285-292). EGFR is activated
when its ligand (either EGF or TGF-.alpha.) binds to the extracellular
domain, resulting in autophosphorylation of the receptor's intracellular
tyrosine kinase domain (S. Cohen et al., 1980, J. Biol. Chem., 255:4834-4842;
A. B. Schreiber et al., 1983, J. Biol. Chem., 258:846-853).
EGFR is the protein product of a growth promoting oncogene, erbB
or ErbB1, that is but one member of a family, i.e., the ERBB family
of protooncogenes, believed to play pivotal roles in the development
and progression of many human cancers. In particular, increased
expression of EGFR has been observed in breast, bladder, lung, head,
neck and stomach cancer as well as glioblastomas. The ERBB family
of oncogenes encodes four, structurally-related transmembrane receptors,
namely, EGFR, HER-2/neu (erbB2), HER-3 (erbB3) and HER-4 (erbB4).
Clinically, ERBB oncogene amplification and/or receptor overexpression
in tumors have been reported to correlate with disease recurrence
and poor patient prognosis, as well as with responsiveness in therapy.
(L. Harris et al., 1999, Int. J. Biol. Markers, 14:8-15; and J.
Mendelsohn and J. Baselga, 2000, Oncogene, 19:6550-6565).
EGFR is composed of three principal domains, namely, the extracellular
domain (ECD), which is glycosylated and contains the ligand-binding
pocket with two cysteine-rich regions; a short transmembrane domain,
and an intracellular domain that has intrinsic tyrosine kinase activity.
The transmembrane region joins the ligand-binding domain to the
intracellular domain. Amino acid and DNA sequence analysis, as well
as studies of nonglycosylated forms of EGFR, indicate that the protein
backbone of EGFR has a mass of 132 kDa, with 1186 amino acid residues
(A. L. Ullrich et al., 1984, Nature, 307:418-425; J. Downward et
al., 1984, Nature, 307:521-527; C. R. Carlin et al., 1986, Mol.
Cell. Biol., 6:257-264; and F. L. V. Mayes and M. D. Waterfield,
1984, The EMBO J., 3:531-537).
The binding of EGF or TGF-.alpha. to EGFR activates a signal transduction
pathway and results in cell proliferation. The dimerization, conformational
changes and internalization of EGFR molecules function to transmit
intracellular signals leading to cell growth regulation (G. Carpenter
and S. Cohen, 1979, Ann. Rev. Biochem., 48:193-216). Genetic alterations
that affect the regulation of growth factor receptor function, or
lead to overexpression of receptor and/or ligand, result in cell
proliferation. In addition, EGFR has been determined to play a role
in cell differentiation, enhancement of cell motility, protein secretion,
neovascularization, invasion, metastasis and resistance of cancer
cells to chemotherapeutic agents and radiation. (M.-J. Oh et al.,
2000, Clin. Cancer Res., 6:4760-4763).
A variety of inhibitors of EGFR have been identified, including
a number already undergoing clinical trials for treatment of various
cancers. For a recent summary, see de Bono, J. S. and Rowinsky,
E. K. (2002), "The ErbB Receptor Family: A Therapeutic Target
For Cancer", Trends in Molecular Medicine, 8, S19-26.
A promising set of targets for therapeutic intervention in the
treatment of cancer includes the members of the HER-kinase axis.
They are frequently upregulated in solid epithelial tumors of, by
way of example, the prostate, lung and breast, and are also upregulated
in glioblastoma tumors. Epidermal growth factor receptor (EGFR)
is a member of the HER-kinase axis, and has been the target of choice
for the development of several different cancer therapies. EGFR
tyrosine kinase inhibitors (EGFR-TKIs) are among these therapies,
since the reversible phosphorylation of tyrosine residues is required
for activation of the EGFR pathway. In other words, EGFR-TKIs block
a cell surface receptor responsible for triggering and/or maintaining
the cell signaling pathway that induces tumor cell growth and division.
Specifically, it is believed that these inhibitors interfere with
the EGFR kinase domain, referred to as HER-1. Among the more promising
EGFR-TKIs are three series of compounds: quinazolines, pyridopyrimidines
and pyrrolopyrimidines.
Two of the more advanced compounds in clinical development include
Gefitinib (compound ZD1839 developed by AstraZeneca UK Ltd.; available
under the tradename IRESSA; hereinafter "IRESSA") and
Erlotinib (compound OSI-774 developed by Genentech, Inc. and OSI
Pharmaceuticals, Inc.; available under the tradename TARCEVA; hereinafter
"TARCEVA"); both have generated encouraging clinical results.
Conventional cancer treatment with both IRESSA and TARCEVA involves
the daily, oral administration of no more than 500 mg of the respective
compounds. In May, 2003, IRESSA became the first of these products
to reach the United States market, when it was approved for the
treatment of advanced non-small cell lung cancer patients.
IRESSA is an orally active quinazoline that functions by directly
inhibiting tyrosine kinase phosphorylation on the EGFR molecule.
It competes for the adenosine triphosphate (ATP) binding site, leading
to suppression of the HER-kinase axis. The exact mechanism of the
IRESSA response is not completely understood, however, studies suggest
that the presence of EGFR is a necessary prerequisite for its action.
A significant limitation in using these compounds is that recipients
thereof may develop a resistance to their therapeutic effects after
they initially respond to therapy, or they may not respond to EGFR-TKIs
to any measurable degree at all. In fact, only 10-15 percent of
advanced non-small cell lung cancer patients respond to EGFR kinase
inhibitors. Thus, a better understanding of the molecular mechanisms
underlying sensitivity to IRESSA and TARCEVA would be extremely
beneficial in targeting therapy to those individuals whom are most
likely to benefit from such therapy.
There is a significant need in the art for a satisfactory treatment
of cancer, and specifically epithelial cell cancers such as lung,
ovarian, breast, brain, colon and prostate cancers, which incorporates
the benefits of TKI therapy and overcoming the non-responsiveness
exhibited by patients. Such a treatment could have a dramatic impact
on the health of individuals, and especially older individuals,
among whom cancer is especially common.
SUMMARY
Tyrosine kinase inhibitor (TKI) therapy such as gefitinib (IRESSA.RTM.)
is not effective in the vast majority of individuals that are affected
with the cancers noted above. The present inventors have surprisingly
discovered that the presence of somatic mutations in the kinase
domain of EGFR substantially increases sensitivity of the EGFR to
TKI such as IRESSA, TARCEVA. For example less than 30% of patients
having such cancer are susceptible to treatment by current TKIs,
whereas greater than 50%, more preferably 60, 70, 80, 90% of patients
having a mutation in the EGFR kinase domain are susceptible. In
addition, these mutations confer increased kinase activity of the
EGFR. Thus, patients having these mutations will likely be responsive
to current tyrosine kinase inhibitor (TKI) therapy, for example,
gefitinib.
Accordingly, the present invention provides a novel method to determine
the likelihood of effectiveness of an epidermal growth factor receptor
(EGFR) targeting treatment in a human patient affected with cancer.
The method comprises detecting the presence or absence of at least
one nucleic acid variance in the kinase domain of the erbB1 gene
of said patient relative to the wildtype erbB1 gene. The presence
of at least one variance indicates that the EGFR targeting treatment
is likely to be effective. Preferably, the nucleic acid variance
increases the kinase activity of the EGFR. The patient can then
be treated with an EGFR targeting treatment. In one embodiment of
the present invention, the EGFR targeting treatment is a tyrosine
kinase inhibitor. In a preferred embodiment, the tyrosine kinase
inhibitor is an anilinoquinazoline. The anilinoquinazoline may be
a synthetic anilinoquinazoline. Preferably, the synthetic anilinoquinazoline
is either gefitinib or erlotinib. In another embodiment, the EGFR
targeting treatment is an irreversible EGFR inhibitor, including
4-dimethylamino-but-2-enoic acid [4-(3-chloro-4-fluoro-phenylamino)-3-cyano-7-ethoxy-quinolin-6-yl]-amide
("EKB-569", sometimes also referred to as "EKI-569",
see for example WO/2005/018677 and Torrance et al., Nature Medicine,
vol. 6, No. 9, September 2000, p. 1024) and/or HKI-272 or HKI-357
(Wyeth; see Greenberger et al., Proc. 11.sup.th NCI EORTC-AACR Symposium
on New Drugs in Cancer Therapy, Clinical Cancer Res. Vol. 6 Supplement,
November 2000, ISSN 1078-0432; in Rabindran et al., Cancer Res.
64: 3958-3965 (2004); Holbro and Hynes, Ann. Rev. Pharm. Tox. 44:195-217
(2004); Tsou et al, j. Med. Chem. 2005, 48, 1107-1131; and Tejpar
et al., J. Clin. Oncol. ASCO Annual Meeting Proc. Vol. 22, No. 14S:
3579 (2004)).
In one embodiment of the present invention, the EGFR is obtained
from a biological sample from a patient with or at risk for developing
cancer. The variance in the kinase domain of EGFR (or the erbB1
gene) effects the conformational structure of the ATP-binding pocket.
Preferably, the variance in the kinase domain of EGFR is an in frame
deletion or a substitution in exon 18, 19, 20 or 21.
In one embodiment, the in frame deletion is in exon 19 of EGFR
(erbB1). The in frame deletion in exon 19 preferably comprises at
deletion of at least amino acids leucine, arginine, glutamic acid
and alanine, at codons 747, 748, 749, and 750. In one embodiment,
the in-frame deletion comprises nucleotides 2235 to 2249 and deletes
amino acids 746 to 750 (the sequence glutamic acid, leucine, arginine,
glutamic acid, and alanine), see Table 2, Table S2, FIG. 2B, FIG.
4A, FIG. 5, SEQ ID NO: 511, FIG. 6C, and FIG. 8C. In another embodiment,
the in-frame deletion comprises nucleotides 2236 to 2250 and deletes
amino acids 746 to 750, see Table S2, FIG. 5, SEQ ID NO: 511, and
FIG. 6C. Alternatively, the in-frame deletion comprises nucleotides
2240 to 2251, see Table 2, FIG. 2C, FIG. 4A, FIG. 5, SEQ ID NO:
511, or nucleotides 2240 to 2257, see Table 2, Table S3A, FIG. 2A,
FIG. 4A, FIG. 5, SEQ ID NO: 511, FIG. 6C, and FIG. 8E. Alternatively,
the in-frame deletion comprises nucleotides 2239 to 2247 together
with a substitution of cytosine for guanine at nucleotide 2248,
see Table S3A and FIG. 8D, or a deletion of nucleotides 2238 to
2255 together with a substitution of thymine for adenine at nucleotide
2237, see Table S3A and FIG. 8F, or a deletion of nucleotides 2254
to 2277, see Table S2 (SEQ ID NO: 437). Alternatively, the in-frame
deletion comprises nucleotides 2239-2250delTTAAGAGAAGCA (SEQ ID
NO: 554); 2251A>C, or 2240-2250delTAAGAGAAGCA (SEQ ID NO: 720),
or 2257-2271delCCGAAAGCCAACAAG (SEQ ID NO: 721), as shown in Table
S3B.
In another embodiment, the substitution is in exon 21 of EGFR.
The substitution in exon 21 comprises at least one amino acid. In
one embodiment, the substitution in exon 21 comprises a substitution
of a guanine for a thymine at nucleotide 2573, see FIG. 4A and FIG.
5, SEQ ID NO: 511. This substitution results in an amino acid substitution,
where the wildtype Leucine is replaced with an Arginine at amino
acid 858, see FIG. 5, Table 2, Table S2, Table S3A, FIG. 2D, FIG.
6A, FIG. 8B, and SEQ ID NO: 512. Alternatively, the substitution
in exon 21 comprises a substitution of an adenine for a thymine
at nucleotide 2582, see FIG. 4A and FIG. 5, SEQ ID NO: 511. This
substitution results in an amino acid substitution, where the wildtype
Leucine is replaced with a Glutamine at amino acid 861, see FIG.
5 (SEQ ID NOS 740-762, respectively, in order of appearance), Table
2 (SEQ ID NOS 730-739, respectively, in order of appearance), FIG.
2E, Table S3B (SEQ ID NOS 554 & 720-729, respectively, in order
of appearance), and SEQ ID NO: 512.
The substitution may also be in exon 18 of EGFR. In one embodiment,
the substitution is in exon 18 is a thymine for a guanine at nucleotide
2155, see FIG. 4A and FIG. 5, SEQ ID NO: 511. This substitution
results in an amino acid substitution, where the wildtype Glycine
is substituted with a Cysteine at codon 719, see FIG. 5, SEQ ID
NO: 512. In another embodiment, the substitution in exon 18 is an
adenine for a guanine at nucleotide 2155 resulting in an amino acid
substitution, where the wildtype Glycine is substituted for a Serine
at codon 719, see Table S2, FIG. 6B, FIG. 8A, FIG. 5, SEQ ID NO:
511 and 512.
In another embodiment, the substitution is an insertion of guanine,
guanine and thymine (GGT) after nucleotide 2316 and before nucleotide
2317 of SEQ ID NO: 511 (2316.sub.--2317 ins GGT). This can also
be described as an insertion of valine (V) at amino acid 772 (P772_H733
insV). Other mutations are shown in Table S3B and include, for example,
and insertion of CAACCCGG after nucleotide 2309 and before nucleotide
2310 of SEQ ID NO 511 and an insertion of GCGTGGACA after nucleotide
2311 and before nucleotide 2312 of SEQ ID NO 511. The substitution
may also be in exon 20 and in one embodiment is a substitution of
AA for GG at nucleotides 2334 and 2335, see Table S3B.
In summary, in preferred embodiments, the nucleic acid variance
of the erbB1 gene is a substitution of a thymine for a guanine or
an adenine for a guanine at nucleotide 2155 of SEQ ID NO 511, a
deletion of nucleotides 2235 to 2249, 2240 to 2251, 2240 to 2257,
2236 to 2250, 2254 to 2277, or 2236 to 2244 of SEQ ID NO 511, an
insertion of nucleotides guanine, guanine, and thymine (GGT) after
nucleotide 2316 and before nucleotide 2317 of SEQ ID NO 511, and
a substitution of a guanine for a thymine at nucleotide 2573 or
an adenine for a thymine at nucleotide 2582 of SEQ ID NO 511.
The detection of the presence or absence of at least one nucleic
acid variance can be determined by amplifying a segment of nucleic
acid encoding the receptor. The segment to be amplified is 1000
nucleotides in length, preferably, 500 nucleotides in length, and
most preferably 100 nucleotides in length or less. The segment to
be amplified can include a plurality of variances.
In another embodiment, the detection of the presence or absence
of at least one variance provides for contacting EGFR nucleic acid
containing a variance site with at least one nucleic acid probe.
The probe preferentially hybridizes with a nucleic acid sequence
including a variance site and containing complementary nucleotide
bases at the variance site under selective hybridization conditions.
Hybridization can be detected with a detectable label.
In yet another embodiment, the detection of the presence or absence
of at least one variance comprises sequencing at least one nucleic
acid sequence and comparing the obtained sequence with the known
erbB1 nucleic acid sequence. Alternatively, the presence or absence
of at least one variance comprises mass spectrometric determination
of at least one nucleic acid sequence.
In a preferred embodiment, the detection of the presence or absence
of at least one nucleic acid variance comprises performing a polymerase
chain reaction (PCR). The erbB1 nucleic acid sequence containing
the hypothetical variance is amplified and the nucleotide sequence
of the amplified nucleic acid is determined. Determining the nucleotide
sequence of the amplified nucleic acid comprises sequencing at least
one nucleic acid segment. Alternatively, amplification products
can analyzed by using any method capable of separating the amplification
products according to their size, including automated and manual
gel electrophoresis and the like.
Alternatively, the detection of the presence or absence of at least
one variance comprises determining the haplotype of a plurality
of variances in a gene.
In another embodiment, the presence or absence of an EGFR variance
can be detected by analyzing the erbB1 gene product (protein). In
this embodiment, a probe that specifically binds to a variant EGFR
is utilized. In a preferred embodiment, the probe is an antibody
that preferentially binds to a variant EGFR. The presence of a variant
EGFR predicts the likelihood of effectiveness of an EGFR targeting
treatment. Alternatively, the probe may be an antibody fragment,
chimeric antibody, humanized antibody or an aptamer.
The present invention further provides a probe which specifically
binds under selective binding conditions to a nucleic acid sequence
comprising at least one nucleic acid variance in the EGFR gene (erbB1).
In one embodiment, the variance is a mutation in the kinase domain
of erbB1 that confers a structural change in the ATP-binding pocket.
The probe of the present invention may comprise a nucleic acid
sequence of about 500 nucleotide bases, preferably about 100 nucleotides
bases, and most preferably about 50 or about 25 nucleotide bases
or fewer in length. The probe may be composed of DNA, RNA, or peptide
nucleic acid (PNA). Furthermore, the probe may contain a detectable
label, such as, for example, a fluorescent or enzymatic label.
The present invention additionally provides a novel method to determine
the likelihood of effectiveness of an epidermal growth factor receptor
(EGFR) targeting treatment in a patient affected with cancer. The
method comprises determining the kinase activity of the EGFR in
a biological sample from a patient. An increase in kinase activity
following stimulation with an EGFR ligand, compared to a normal
control, indicates that the EGFR targeting treatment is likely to
be effective.
The present invention further provides a novel method for treating
a patient affected with or at risk for developing cancer. The method
involves determining whether the kinase domain of the EGFR of a
patient contains at least one nucleic acid variance. Preferably,
the EGFR is located at the site of the tumor or cancer and the nucleic
acid variance is somatic. The presence of such a variance indicates
that an EGFR targeted treatment will be effective. If the variance
is present, the tyrosine kinase inhibitor is administered to the
patient.
As above, the tyrosine kinase inhibitor administered to an identified
patient may be an anilinoquinazoline or an irreversible tyrosine
kinase inhibitor, such as for example, EKB-569, HKI-272 and/or HKI-357
(Wyeth). Preferably, the anilinoquinazoline is a synthetic anilinoquinazoline
and most preferably the synthetic anilinoquinazoline is gefitinib
and erlotinib.
The cancer to be treated by the methods of the present invention
include, for example, but are not limited to, gastrointestinal cancer,
prostate cancer, ovarian cancer, breast cancer, head and neck cancer,
lung cancer, non-small cell lung cancer, cancer of the nervous system,
kidney cancer, retina cancer, skin cancer, liver cancer, pancreatic
cancer, genital-urinary cancer and bladder cancer. In a preferred
embodiment, the cancer is non-small cell lung cancer.
A kit for implementing the PCR methods of the present invention
is also encompassed. The kit includes at least one degenerate primer
pair designed to anneal to nucleic acid regions bordering the genes
that encode for the ATP-binding pocket of the EGFR kinase domain.
Additionally, the kit contains the products and reagents required
to carry out PCR amplification, and instructions.
In a preferred embodiment, the primer pairs contained within the
kit are selected from the group consisting of SEQ ID NO: 505, SEQ
ID NO: 506, SEQ ID NO: 507, and SEQ ID NO: 508. Also preferred are
the primers listed in Table 6 and 7 in the examples.
In yet another embodiment, the present invention discloses a method
for selecting a compound that inhibits the catalytic kinase activity
of a variant epidermal growth factor receptor (EGFR). As a first
step, a variant EGFR is contacted with a potential compound. The
resultant kinase activity of the variant EGFR is then detected and
a compound is selected that inhibits the kinase activity of the
variant EGFR. In one embodiment, the variant EGFR is contained within
a cell. The method can also be used to select a compound that inhibits
the kinase activity of a variant EGFR having a secondary mutation
in the kinase domain that confers resistance to a TKI, e.g., gefitinib
or erlotinib.
In one embodiment, the variant EGFR is labeled. In another embodiment,
the EGFR is bound to a solid support. In a preferred embodiment,
the solid support is a protein chip.
In yet another embodiment of the present invention, a pharmaceutical
composition that inhibits the catalytic kinase activity of a variant
epidermal growth factor receptor (EGFR) is disclosed. The compound
that inhibits the catalytic kinase activity of a variant EGFR is
selected from the group consisting of an antibody, antibody fragment,
small molecule, peptide, protein, antisense nucleic acid, ribozyme,
PNA, siRNA, oligonucleotide aptamer, and peptide aptamer.
A method for treating a patient having an EGFR mediated disease
is also disclosed. In accordance with the method, the patient is
administered the pharmaceutical composition that inhibits the catalytic
kinase activity of a variant epidermal growth factor receptor (EGFR).
In one embodiment, the EGFR mediated disease is cancer. In a preferred
embodiment, the cancer is of epithelial origin. For example, the
cancer is gastrointestinal cancer, prostate cancer, ovarian cancer,
breast cancer, head and neck cancer, lung cancer, non-small cell
lung cancer, cancer of the nervous system, kidney cancer, retina
cancer, skin cancer, liver cancer, pancreatic cancer, genital-urinary
cancer and bladder cancer. In a preferred embodiment, the cancer
is non-small cell lung cancer.
In another embodiment, a method for predicting the acquisition
of secondary mutations (or selecting for mutations) in the kinase
domain of the erbB1 gene is disclosed. A cell expressing a variant
form of the erbB1 gene is contacted with an effective, yet sub-lethal
dose of a tyrosine kinase inhibitor. Cells that are resistant to
a growth arrest effect of the tyrosine kinase inhibitor are selected
and the erbB1 nucleic acid is analyzed for the presence of additional
mutations in the erbB1 kinase domain. In one embodiment, the cell
is in vitro. In another embodiment, the cell is obtained from a
transgenic animal. In one embodiment, the transgenic animal is a
mouse. In this mouse model, cells to be studied are obtained from
a tumor biopsy. Cells containing a secondary mutation in the erbB1
kinase domain selected by the present invention can be used in the
above methods to select a compound that inhibits the kinase activity
of the variant EGFR having a secondary mutation in the kinase domain.
In an alternative embodiment for predicting the acquisition of
secondary mutations in the kinase domain of the erbB1 gene, cells
expressing a variant form of the erbB1 gene are first contacted
with an effective amount of a mutagenizing agent. The mutagenizing
is, for example, ethyl methanesulfonate (EMS), N-ethyl-N-nitrosourea
(ENU), N-methyl-N-nitrosourea (MNU), phocarbaxine hydrochloride
(Prc), methyl methanesulfonate (MeMS), chlorambucil (Chl), melphalan,
porcarbazine hydrochloride, cyclophosphamide (Cp), diethyl sulfate
(Et.sub.2SO.sub.4), acrylamide monomer (AA), triethylene melamin
(TEM), nitrogen mustard, vincristine, dimethylnitrosamine, N-methyl-N'-nitro-Nitrosoguanidine
(MNNG), 7,12 dimethylbenz(a)anthracene (DMBA), ethylene oxide, hexamethylphosphoramide,
bisulfan, or ethyl methanesulforate (EtMs). The cell is then contacted
with an effective, yet sub-lethal dose of a tyrosine kinase inhibitor.
Cells that are resistant to a growth arrest effect of the tyrosine
kinase inhibitor are selected and the erbB1 nucleic acid is analyzed
for the presence of additional mutations in the erbB1 kinase domain.
BRIEF DESCRIPTION OF THE FIGURES
FIGS. 1A-1B show a representative illustration of Gefitinib response
in refractory non-small cell lung cancer (NSCLC). Chest CT scan
of case 6 (Table 1), demonstrating (FIG. 1A) a large mass in the
right lung before treatment with gefitinib, and (FIG. 1B) marked
improvement six weeks after Gefitinib was initiated.
FIGS. 2A-2F show EGFR mutations in Gefitinib-responsive tumors.
FIGS. 2A-2C show nucleotide sequence of the EGFR gene in tumor
specimens with heterozygous in-frame deletions within the kinase
domain (double peaks) (SEQ ID NOS 643, 644 and 690-699, respectively,
in order of appearance). Tracings in both sense and antisense directions
are shown to demonstrate the two breakpoints of the deletion; wild-type
nucleotide sequence is shown in capital letters, and the mutant
sequence is in lowercase letters. The 5' breakpoint of the delL747-T751insS
mutation is preceded by a T to C substitution that does not alter
the encoded amino acid.
FIG. 2D and FIG. 2E show heterozygous missense mutations (arrows)
resulting in amino acid substitutions within the tyrosine kinase
domain (SEQ ID NOS 701 & 703). The double peaks represent two
nucleotides at the site of heterozygous mutations. For comparison,
the corresponding wild-type sequence is also shown (SEQ ID NOS 700
& 702).
FIG. 2F is a schematic representation of dimerized EGFR molecules
bound by the EGF ligand. The extracellular domain (containing two
receptor ligand [L]-domains and a furin-like domain), transmembrane
region, and the cytoplasmic domain (containing the catalytic kinase
domain) are highlighted. The position of tyrosine.sup.1068 (Y-1068),
a site of autophosphorylation used as a marker of receptor activation,
is indicated, along with downstream effectors activated by EGFR
autophosphorylation (STAT3, MAP Kinase (MAPK), and AKT). The location
of tumor-associated mutations, all within the tyrosine kinase domain,
is shown.
FIGS. 3A-3D demonstrate enhanced EGF-dependent activation of mutant
EGFR and increased sensitivity of mutant EGFR to Gefitinib.
FIG. 3A shows a time course of ligand-induced activation of the
delL747-P753insS and L858R mutants, compared with wild type EGFR,
following addition of EGF to serum starved cells. EGFR autophosphorylation
is used as a marker of receptor activation, using Western blotting
with an antibody that specifically recognizes the phosphorylated
tyrosine.sup.1068 residue of EGFR (left panel), compared with the
total levels of EGFR expressed in Cos-7 cells (control; right panel).
Autophosphorylation of EGFR is measured at intervals following addition
of EGF (10 ng/ml).
FIG. 3B is a graphical representation of EGF-induction of wild-type
and mutant receptor phosphorylation (see panel A). Autoradiographs
from three independent experiments were quantified using the NIH
image software; intensity of EGFR phosphorylation is normalized
to total protein expression, and shown as percent activation of
the receptor, with standard deviation.
FIG. 3C shows a dose-dependent inhibition of EGFR activation by
Gefitinib. Autophosphorylation of EGFR tyrosine.sup.1068 is demonstrated
by Western blotting analysis of Cos-7 cells expressing wild-type
or mutant receptors, and stimulated with 100 ng/ml of EGF for 30
min. Cells were untreated (U) or pretreated for 3 hrs with increasing
concentrations of Gefitinib as shown (left panel). Total amounts
of EGFR protein expressed are shown as control (right panel).
FIG. 3D shows the quantification of results from two experiments
described for panel 3C (NIH image software). Concentrations of phosphorylated
EGFR were normalized to protein expression levels and expressed
as percent activation of the receptor.
FIGS. 4A-4C demonstrate clustering of mutations at critical sites
within the ATP-binding pocket of EGFR.
FIG. 4A shows the position of overlapping in-frame deletions in
exon 19 and missense mutations in exon 21 of the EGFR gene, in multiple
cases of NSCLC (SEQ ID NOS 495-504 (DNA)). Partial nucleotide sequence
is shown for each exon, with deletions marked by dashed lines and
missense mutations highlighted and underlined; the wild-type EGFR
nucleotide and amino acid sequences are shown (SEQ ID NOS 493 &
494 (DNA) & 509-510 (amino acid)).
FIG. 4B shows the tridimensional structure of the EGFR ATP cleft
flanked by the amino (N) and carboxy (C) lobes of the kinase domain
(coordinates derived from PDB 1M14, and displayed using Cn3D software).
The inhibitor, representing Gefitinib, is pictured occupying the
ATP cleft. The locations of the two missense mutations are shown,
within the activating loop of the kinase; the three in-frame deletions
are all present within another loop, which flanks the ATP cleft.
FIG. 4C is a close-up of the EGFR kinase domain, showing the critical
amino acid residues implicated in binding to either ATP or to the
inhibitor. Specifically, 4-anilinoquinazoline compounds such as
gefitinib inhibit catalysis by occupying the ATP-binding site, where
they form hydrogen bonds with methionine.sup.793 (M793) and cysteine.sup.775
(C775) residues, whereas their anilino ring is close to methionine.sup.766
(M766), lysine.sup.745 (K745), and leucine.sup.788 (L788) residues.
In-frame deletions within the loop that is targeted by mutations
are predicted to alter the position of these amino acids relative
to the inhibitor. Mutated residues are shown within the activation
loop of the tyrosine kinase.
FIG. 5 shows the nucleotide and amino acid sequence of the erbB1
gene. The amino acids are depicted as single letters, known to those
of skill in the art. Nucleotide variances in the kinase domain are
highlighted by patient number, see Table 2. SEQ ID NO: 511 includes
nucleotides 1 through 3633. SEQ ID NO: 512 includes amino acids
1 through 1210.
FIGS. 6A-6C: Sequence alignment of selected regions within the
EGFR and B-Raf kinase domains. Depiction of EGFR mutations in human
NSCLC. EGFR (gb:X00588;) mutations in NSCLC tumors are highlighted
in gray. B-Raf (gb:M95712) mutations in multiple tumor types (5)
are highlighted in black. Asterisks denote residues conserved between
EGFR and B-Raf. FIG. 6A depicts L858R mutations in the activation
loop (SEQ ID NOS 477-479). FIG. 6B depicts the G719S mutant in the
P-loop (SEQ ID NOS 480-482). FIG. 6C depicts deletion mutants in
EGFR exon 19 (SEQ ID NOS 483-489).
FIG. 7: Positions of missense mutations G719S and L858R and the
Del-1 deletion in the three-dimensional structure of the EGFR kinase
domain. The activation loop is shown in yellow, the P-loop is in
blue and the C-lobe and N-lobe are as indicated. The residues targeted
by mutation or deletion are highlighted in red. The Del-1 mutation
targets the residues ELREA in codons 746 to 750. The mutations are
located in highly conserved regions within kinases and are found
in the p-loop and activation loop, which surround the region where
ATP and also gefitinib and erlotinib are predicted to bind.
FIGS. 8A-8F. Representative chromatograms of EGFR DNA from normal
tissue and from tumor tissues. The locations of the identified mutations
are as follows. FIG. 8A depicts the Exon 18 Kinase domain P loop
(SEQ ID NOS 704-705). FIG. 8B depicts the Exon 21 Kinase domain
A-loop (SEQ ID NOS 706-707). FIG. 8C depicts the Exon 19 Kinase
domain Del-1 (SEQ ID NOS 708-710). FIG. 8D depicts the Exon 19 Kinase
domain Del-3 (SEQ ID NOS 711-713). FIG. 8E depicts the Exon 19 Kinase
domain Del-4 (SEQ ID NOS 714-716). FIG. 8F depicts the Exon 19 Kinase
domain Del-5 (SEQ ID NOS 717-719).
FIG. 9: Sequence alignment of the EGFR and BCR-ABL polypeptides
and the location of residues conferring a drug resistant phenotype.
The EGFR polypeptide (SEQ ID NO:492) encoded by the nucleotide sequence
disclosed in GenBank accno. NM.sub.--005228 and the BCR-ABL polypeptide
(SEQ ID NO:491) encoded by the nucleotide sequence disclosed in
GenBank accno. M14752 are aligned and conserved residues are shaded.
BCR-ABL mutations conferring resistance to the tyrosine kinase inhibitor
imatinib (ST1571, Glivec/Gleevec) are denoted by asterisks.
FIG. 10 shows the decision making process for patient with metastatic
NSCLC undergoing EGFR testing.
FIG. 11 shows a diagram of EGFR exons 18-24 (not to scale). Arrows
deptict the location of identified mutations. Astericks denote the
number of patients with mutations at each location. The blow-up
diagram depicts the overlap of the exon 19 deletions, and the number
of patients (n) with each deletion (nucleotides 2233-2277 of SEQ
ID NO: 511 and residues 745-759 of SEQ ID NO: 512). Note that these
are the results are not meant to be inclusive of all the EGFR mutations
to date.
DETAILED DESCRIPTION
The present invention provides a novel method to determine the
likelihood of effectiveness of an epidermal growth factor receptor
(EGFR) targeting treatment in a patient affected with cancer. The
method comprises detecting the presence or absence of at least one
nucleic acid variance in the kinase domain of the erbB1 gene of
said patient. The presence of at least one variance indicates that
the EGFR targeting treatment is likely to be effective. Preferably,
the nucleic acid variance increases the kinase activity of the EGFR.
The patient can then be treated with an EGFR targeting treatment.
In one embodiment of the present invention, the EGFR targeting treatment
is a tyrosine kinase inhibitor. In a preferred embodiment, the tyrosine
kinase inhibitor is an anilinoquinazoline. The anilinoquinazoline
may be a synthetic anilinoquinazoline. Preferably, the synthetic
anilinoquinazoline is either gefitinib or erlotinib.
Definitions:
The terms "ErbB1", "epidermal growth factor receptor"
and "EGFR" are used interchangeably herein and refer to
native sequence EGFR as disclosed, for example, in Carpenter et
al. Ann. Rev. Biochem. 56:881-914 (1987), including variants thereof
(e.g. a deletion mutant EGFR as in Humphrey et al. PNAS (USA) 87:4207-4211
(1990)). erbB1 refers to the gene encoding the EGFR protein product.
The term "kinase activity increasing nucleic acid variance"
as used herein refers to a variance (i.e. mutation) in the nucleotide
sequence of a gene that results in an increased kinase activity.
The increased kinase activity is a direct result of the variance
in the nucleic acid and is associated with the protein for which
the gene encodes.
The term "drug" or "compound" as used herein
refers to a chemical entity or biological product, or combination
of chemical entities or biological products, administered to a person
to treat or prevent or control a disease or condition. The chemical
entity or biological product is preferably, but not necessarily
a low molecular weight compound, but may also be a larger compound,
for example, an oligomer of nucleic acids, amino acids, or carbohydrates
including without limitation proteins, oligonucleotides, ribozymes,
DNAzymes, glycoproteins, siRNAs, lipoproteins, aptamers, and modifications
and combinations thereof.
The term "genotype" in the context of this invention
refers to the particular allelic form of a gene, which can be defined
by the particular nucleotide(s) present in a nucleic acid sequence
at a particular site(s).
The terms "variant form of a gene", "form of a gene",
or "allele" refer to one specific form of a gene in a
population, the specific form differing from other forms of the
same gene in the sequence of at least one, and frequently more than
one, variant sites within the sequence of the gene. The sequences
at these variant sites that differ between different alleles of
the gene are termed "gene sequence variances" or "variances"
or "variants". Other terms known in the art to be equivalent
include mutation and polymorphism, although mutation is often used
to refer to an allele associated with a deleterious phenotype. In
preferred aspects of this invention, the variances are selected
from the group consisting of the variances listed in the variance
tables herein.
In the context of this invention, the term "probe" refers
to a molecule which can detectably distinguish between target molecules
differing in structure. Detection can be accomplished in a variety
of different ways depending on the type of probe used and the type
of target molecule. Thus, for example, detection may be based on
discrimination of activity levels of the target molecule, but preferably
is based on detection of specific binding. Examples of such specific
binding include antibody binding and nucleic acid probe hybridization.
Thus, for example, probes can include enzyme substrates, antibodies
and antibody fragments, and preferably nucleic acid hybridization
probes.
As used herein, the terms "effective" and "effectiveness"
includes both pharmacological effectiveness and physiological safety.
Pharmacological effectiveness refers to the ability of the treatment
to result in a desired biological effect in the patient. Physiological
safety refers to the level of toxicity, or other adverse physiological
effects at the cellular, organ and/or organism level (often referred
to as side-effects) resulting from administration of the treatment.
"Less effective" means that the treatment results in a
therapeutically significant lower level of pharmacological effectiveness
and/or a therapeutically greater level of adverse physiological
effects.
The term "primer", as used herein, refers to an oligonucleotide
which is capable of acting as a point of initiation of polynucleotide
synthesis along a complementary strand when placed under conditions
in which synthesis of a primer extension product which is complementary
to a polynucleotide is catalyzed. Such conditions include the presence
of four different nucleotide triphosphates or nucleoside analogs
and one or more agents for polymerization such as DNA polymerase
and/or reverse transcriptase, in an appropriate buffer ("buffer"
includes substituents which are cofactors, or which affect pH, ionic
strength, etc.), and at a suitable temperature. A primer must be
sufficiently long to prime the synthesis of extension products in
the presence of an agent for polymerase. A typical primer contains
at least about 5 nucleotides in length of a sequence substantially
complementary to the target sequence, but somewhat longer primers
are preferred. Usually primers contain about 15-26 nucleotides,
but longer primers may also be employed.
A primer will always contain a sequence substantially complementary
to the target sequence, that is the specific sequence to be amplified,
to which it can anneal. A primer may, optionally, also comprise
a promoter sequence. The term "promoter sequence" defines
a single strand of a nucleic acid sequence that is specifically
recognized by an RNA polymerase that binds to a recognized sequence
and initiates the process of transcription by which an RNA transcript
is produced. In principle, any promoter sequence may be employed
for which there is a known and available polymerase that is capable
of recognizing the initiation sequence. Known and useful promoters
are those that are recognized by certain bacteriophage polymerases,
such as bacteriophage T3, T7 or SP6.
A "microarray" is a linear or two-dimensional array of
preferably discrete regions, each having a defined area, formed
on the surface of a solid support. The density of the discrete regions
on a microarray is determined by the total numbers of target polynucleotides
to be detected on the surface of a single solid phase support, preferably
at least about 50/cm.sup.2, more preferably at least about 100/cm.sup.2,
even more preferably at least about 500/cm.sup.2, and still more
preferably at least about 1,000/cm.sup.2. As used herein, a DNA
microarray is an array of oligonucleotide primers placed on a chip
or other surfaces used to amplify or clone target polynucleotides.
Since the position of each particular group of primers in the array
is known, the identities of the target polynucleotides can be determined
based on their binding to a particular position in the microarray.
The term "label" refers to a composition capable of producing
a detectable signal indicative of the presence of the target polynucleotide
in an assay sample. Suitable labels include radioisotopes, nucleotide
chromophores, enzymes, substrates, fluorescent molecules, chemiluminescent
moieties, magnetic particles, bioluminescent moieties, and the like.
As such, a label is any composition detectable by spectroscopic,
photochemical, biochemical, immunochemical, electrical, optical
or chemical means.
The term "support" refers to conventional supports such
as beads, particles, dipsticks, fibers, filters, membranes and silane
or silicate supports such as glass slides.
The term "amplify" is used in the broad sense to mean
creating an amplification product which may include, for example,
additional target molecules, or target-like molecules or molecules
complementary to the target molecule, which molecules are created
by virtue of the presence of the target molecule in the sample.
In the situation where the target is a nucleic acid, an amplification
product can be made enzymatically with DNA or RNA polymerases or
reverse transcriptases.
As used herein, a "biological sample" refers to a sample
of tissue or fluid isolated from an individual, including but not
limited to, for example, blood, plasma, serum, tumor biopsy, urine,
stool, sputum, spinal fluid, pleural fluid, nipple aspirates, lymph
fluid, the external sections of the skin, respiratory, intestinal,
and genitourinary tracts, tears, saliva, milk, cells (including
but not limited to blood cells), tumors, organs, and also samples
of in vitro cell culture constituent. In a preferred embodiment,
the sample is from a resection, bronchoscopic biopsy, or core needle
biopsy of a primary or metastatic tumor, or a cellblock from pleural
fluid. In addition, fine needle aspirate samples are used. Samples
may be either paraffin-embedded or frozen tissue.
The term "antibody" is meant to be an immunoglobulin
protein that is capable of binding an antigen. Antibody as used
herein is meant to include antibody fragments, e.g. F(ab')2, Fab',
Fab, capable of binding the antigen or antigenic fragment of interest.
Preferably, the binding of the antibody to the antigen inhibits
the activity of a variant form of EGFR.
The term "humanized antibody" is used herein to describe
complete antibody molecules, i.e. composed of two complete light
chains and two complete heavy chains, as well as antibodies consisting
only of antibody fragments, e.g. Fab, Fab', F (ab') 2, and Fv, wherein
the CDRs are derived from a non-human source and the remaining portion
of the Ig molecule or fragment thereof is derived from a human antibody,
preferably produced from a nucleic acid sequence encoding a human
antibody.
The terms "human antibody" and "humanized antibody"
are used herein to describe an antibody of which all portions of
the antibody molecule are derived from a nucleic acid sequence encoding
a human antibody. Such human antibodies are most desirable for use
in antibody therapies, as such antibodies would elicit little or
no immune response in the human patient.
The term "chimeric antibody" is used herein to describe
an antibody molecule as well as antibody fragments, as described
above in the definition of the term "humanized antibody."
The term "chimeric antibody" encompasses humanized antibodies.
Chimeric antibodies have at least one portion of a heavy or light
chain amino acid sequence derived from a first mammalian species
and another portion of the heavy or light chain amino acid sequence
derived from a second, different mammalian species.
Preferably, the variable region is derived from a non-human mammalian
species and the constant region is derived from a human species.
Specifically, the chimeric antibody is preferably produced from
a 9 nucleotide sequence from a non-human mammal encoding a variable
region and a nucleotide sequence from a human encoding a constant
region of an antibody.
Table 2 is a partial list of DNA sequence variances in the kinase
domain of erbB1 relevant to the methods described in the present
invention. These variances were identified by the inventors in studies
of biological samples from patients with NSCLC who responded to
gefitinib and patients with no exposure to gefitinb.
Nucleic acid molecules can be isolated from a particular biological
sample using any of a number of procedures, which are well-known
in the art, the particular isolation procedure chosen being appropriate
for the particular biological sample. For example, freeze-thaw and
alkaline lysis procedures can be useful for obtaining nucleic acid
molecules from solid materials; heat and alkaline lysis procedures
can be useful for obtaining nucleic acid molecules from urine; and
proteinase K extraction can be used to obtain nucleic acid from
blood (Rolff, A et al. PCR: Clinical Diagnostics and Research, Springer
(1994).
Detection Methods
Determining the presence or absence of a particular variance or
plurality of variances in the kinase domain of the erbB1 gene in
a patient with or at risk for developing cancer can be performed
in a variety of ways. Such tests are commonly performed using DNA
or RNA collected from biological samples, e.g., tissue biopsies,
urine, stool, sputum, blood, cells, tissue scrapings, breast aspirates
or other cellular materials, and can be performed by a variety of
methods including, but not limited to, PCR, hybridization with allele-specific
probes, enzymatic mutation detection, chemical cleavage of mismatches,
mass spectrometry or DNA sequencing, including minisequencing. In
particular embodiments, hybridization with allele specific probes
can be conducted in two formats: (1) allele specific oligonucleotides
bound to a solid phase (glass, silicon, nylon membranes) and the
labeled sample in solution, as in many DNA chip applications, or
(2) bound sample (often cloned DNA or PCR amplified DNA) and labeled
oligonucleotides in solution (either allele specific or short so
as to allow sequencing by hybridization). Diagnostic tests may involve
a panel of variances, often on a solid support, which enables the
simultaneous determination of more than one variance.
In another aspect, determining the presence of at least one kinase
activity increasing nucleic acid variance in the erbB1 gene may
entail a haplotyping test. Methods of determining haplotypes are
known to those of skill in the art, as for example, in WO 00/04194.
Preferably, the determination of the presence or absence of a kinase
activity increasing nucleic acid variance involves determining the
sequence of the variance site or sites by methods such as polymerase
chain reaction (PCR). Alternatively, the determination of the presence
or absence of a kinase activity increasing nucleic acid variance
may encompass chain terminating DNA sequencing or minisequencing,
oligonucleotide hybridization or mass spectrometry.
The methods of the present invention may be used to predict the
likelihood of effectiveness (or lack of effectiveness) of an EGFR
targeting treatment in a patient affected with or at risk for developing
cancer. Preferably, cancers include cancer of epithelial origin,
including, but are not limited to, gastrointestinal cancer, prostate
cancer, ovarian cancer, breast cancer, head and neck cancer, lung
cancer, non-small cell lung cancer, cancer of the nervous system,
kidney cancer, retina cancer, skin cancer, liver cancer, pancreatic
cancer, genital-urinary cancer and bladder cancer. In a preferred
embodiment, the cancer is non-small cell lung cancer.
The present invention generally concerns the identification of
variances in the kinase domain of the erbB1 gene which are indicative
of the effectiveness of an EGFR targeting treatment in a patient
with or at risk for developing cancer. Additionally, the identification
of specific variances in the kinase domain of EGFR, in effect, can
be used as a diagnostic or prognostic test. For example, the presence
of at least one variance in the kinase domain of erbB1 indicates
that a patient will likely benefit from treatment with an EGFR targeting
compound, such as, for example, a tyrosine kinase inhibitor.
Methods for diagnostic tests are well known in the art and disclosed
in patent application WO 00/04194, incorporated herein by reference.
In an exemplary method, the diagnostic test comprises amplifying
a segment of DNA or RNA (generally after converting the RNA to cDNA)
spanning one or more known variances in the kinase domain of the
erbB1 gene sequence. This amplified segment is then sequenced and/or
subjected to polyacrylamide gel electrophoresis in order to identify
nucleic acid variances in the amplified segment.
PCR
In one embodiment, the invention provides a method of screening
for variants in the kinase domain of the erbB1 gene in a test biological
sample by PCR or, alternatively, in a ligation chain reaction (LCR)
(see, e.g., Landegran, et al., 1988. Science 241: 1077-1080; and
Nakazawa, et al., 1994. Proc. Natl. Acad. Sci. USA 91: 360-364),
the latter of which can be particularly useful for detecting point
mutations in the EGFR-gene (see, Abravaya, et al., 1995. Nucl. Acids
Res. 23: 675-682). The method comprises the steps of designing degenerate
primers for amplifying the target sequence, the primers corresponding
to one or more conserved regions of the gene, amplifying reaction
with the primers using, as a template, a DNA or cDNA obtained from
a test biological sample and analyzing the PCR products. Comparison
of the PCR products of the test biological sample to a control sample
indicates variances in the test biological sample. The change can
be either and absence or presence of a nucleic acid variance in
the test biological sample.
Alternative amplification methods include: self sustained sequence
replication (see, Guatelli, et al., 1990. Proc. Natl. Acad. Sci.
USA 87: 1874-1878), transcriptional amplification system (see, Kwoh,
et al., 1989. Proc. Natl. Acad. Sci. USA 86: 1173-1177); Qb Replicase
(see, Lizardi, et al, 1988. BioTechnology 6: 1197), or any other
nucleic acid amplification method, followed by the detection of
the amplified molecules using techniques well known to those of
skill in the art. These detection schemes are especially useful
for the detection of nucleic acid molecules if such molecules are
present in very low numbers.
Primers useful according to the present invention are designed
using amino acid sequences of the protein or nucleic acid sequences
of the kinase domain of the erbB1 gene as a guide, e.g. SEQ ID NO:
493, SEQ ID NO: 494, SEQ ID NO: 509, and SEQ ID NO: 510. The primers
are designed in the homologous regions of the gene wherein at least
two regions of homology are separated by a divergent region of variable
sequence, the sequence being variable either in length or nucleic
acid sequence.
For example, the identical or highly. homologous, preferably at
least 80%-85% more preferably at least 90-99% homologous amino acid
sequence of at least about 6, preferably at least 8-10 consecutive
amino acids. Most preferably, the amino acid sequence is 100% identical.
Forward and reverse primers are designed based upon the maintenance
of codon degeneracy and the representation of the various amino
acids at a given position among the known gene family members. Degree
of homology as referred to herein is based upon analysis of an amino
acid sequence using a standard sequence comparison software, such
as protein-BLAST using the default settings.
Table 3 below represents the usage of degenerate codes and their
standard symbols:
TABLE-US-00001 T C A G T TTT Phe (F) TCT Ser (S) TAT Tyr (Y) TGT
Cys (C) TTC Phe (F) TCC Ser (S) TAC TGC TTA Leu (L) TCA Ser (S)
TAA Ter TGA Ter TTG Leu (L) TCG Ser (S) TAG Ter TGG Trp (W) C CTT
Leu (L) CCT Pro (P) CAT His (H) CGT Arg (R) CTC Leu (L) CCC Pro
(P) CAC His (H) CGC Arg (R) CTA Leu (L) CCA Pro (P) CAA Gln (Q)
CGA Arg (R) CTG Leu (L) CCG Pro (P) CAG Gln (Q) CGG Arg (R) A ATT
Ile (I) ACT Thr (T) AAT Asn (N) AGT Ser (S) ATC Ile (I) ACC Thr
(T) AAC Asn (N) AGC Ser (S) ATA Ile (I) ACA Thr (T) AAA Lys (K)
AGA Arg (R) ATG Met (M) ACG Thr (T) AAG Lys (K) AGG Arg (R) G GTT
Val (V) GCT Ala (A) GAT Asp (D) GGT Gly (G) GTC Val (V) GCC Ala
(A) GAC Asp (D) GGC Gly (G) GTA Val (V) GCA Ala (A) GAA Glu (E)
GGA Gly (G) GTG Val (V) GCG Ala (A) GAG Glu (E) GGG Gly (G)
Preferably any 6-fold degenerate codons such as L, R and S are
avoided since in practice they will introduce higher than 6-fold
degeneracy. In the case of L, TTR and CTN are compromised YTN (8-fold
degeneracy), in the case of R, CGN and AGR compromises at MGN (8-fold
degeneracy), and finally S, TCN and AGY which can be compromised
to WSN (16-fold degeneracy). In all three cases on 6 of these will
match the target sequence. To avoid this loss of specificity, it
is preferable to avoid these regions, or to make two populations,
each with the alternative degenerate codon, e.g. for S include TCN
in one pool, and AGY in the other.
Primers may be designed using a number of available computer programs,
including, but not limited to Oligo Analyzer3.0; Oligo Calculator
NetPrimer; Methprimer; Primer3: WebPrimer, PrimerFinder; Primer9;
Oligo2002; Pride or GenomePride; Oligos; and Codehop.
Primers may be labeled using labels known to one skilled in the
art. Such labels include, but are not limited to radioactive, fluorescent,
dye, and enzymatic labels.
Analysis of amplification products can be performed using any method
capable of separating the amplification products according to their
size, including automated and manual gel electrophoresis, mass spectrometry,
and the like.
Alternatively, the amplification products can be separated using
sequence differences, using SSCP, DGGE, TGGE, chemical cleavage
or restriction fragment polymorphisms as well as hybridization to,
for example, a nucleic acid arrays.
The methods of nucleic acid isolation, amplification and analysis
are routine for one skilled in the art and examples of protocols
can be found, for example, in the Molecular Cloning: A Laboratory
Manual (3-Volume Set) Ed. Joseph Sambrook, David W. Russel, and
Joe Sambrook, Cold Spring Harbor Laboratory; 3rd edition (Jan. 15,
2001), ISBN: 0879695773. Particularly useful protocol source for
methods used in PCR amplification is PCR (Basics: From Background
to Bench) by M. J. McPherson, S. G. Moller, R. Beynon, C. Howe,
Springer Verlag; 1st edition (Oct. 15, 2000), ISBN: 0387916008.
Preferably, exons 19 and 21 of human EGFR are amplified by the
polymerase chain reaction (PCR) using the following primers: Exon19
sense primer, 5'-GCAATATCAGCCTTAGGTGCGGCTC-3' (SEQ ID NO: 505);
Exon 19 antisense primer, 5'-CATAGAA AGTGAACATTTAGGATGTG-3' (SEQ
ID NO: 506); Exon 21 sense primer, 5'-CTAACGTTCG CCAGCCATAAGTCC-3'
(SEQ ID NO: 507); and Exon21 antisense primer, 5'-GCTGCGAGCTCACCCAG
AATGTCTGG-3' (SEQ ID NO: 508).
In an alternative embodiment, mutations in a EGFR gene from a sample
cell can be identified by alterations in restriction enzyme cleavage
patterns. For example, sample and control DNA is isolated, amplified
(optionally), digested with one or more restriction endonucleases,
and fragment length sizes are determined by gel electrophoresis
and compared. Differences in fragment length sizes between sample
and control DNA indicates mutations in the sample DNA. Moreover,
the use of sequence specific ribozymes (see, e.g., U.S. Pat. No.
5,493,531) can be used to score for the presence of specific mutations
by development or loss of a ribozyme cleavage site.
Other methods for detecting mutations in the EGFR gene include
methods in which protection from cleavage agents is used to detect
mismatched bases in RNA/RNA or RNA/DNA heteroduplexes. See, e.g.,
Myers, et al., 1985. Science 230: 1242. In general, the art technique
of "mismatch cleavage" starts by providing heteroduplexes
of formed by hybridizing (labeled) RNA or DNA containing the wild-type
EGFR sequence with potentially mutant RNA or DNA obtained from a
tissue sample. The double-stranded duplexes are treated with an
agent that cleaves single-stranded regions of the duplex such as
which will exist due to basepair mismatches between the control
and sample strands. For instance, RNA/DNA duplexes can be treated
with RNase and DNA/DNA hybrids treated with S1 nuclease to enzymatically
digesting the mismatched regions. In other embodiments, either DNA/DNA
or RNA/DNA duplexes can be treated with hydroxylamine or osmium
tetroxide and with piperidine in order to digest mismatched regions.
After digestion of the mismatched regions, the resulting material
is then separated by size on denaturing polyacrylamide gels to determine
the site of mutation. See, e.g., Cotton, et al., 1988. Proc. Natl.
Acad. Sci. USA 85: 4397; Saleeba, et al., 1992. Methods Enzymol.
217: 286-295. In an embodiment, the control DNA or RNA can be labeled
for detection.
In still another embodiment, the mismatch cleavage reaction employs
one or more proteins that recognize mismatched base pairs in double-stranded
DNA (so called "DNA mismatch repair" enzymes) in defined
systems for detecting and mapping point mutations in EGFR cDNAs
obtained from samples of cells. For example, the mutY enzyme of
E. coli cleaves A at G/A mismatches and the thymidine DNA glycosylase
from HeLa cells cleaves T at G/T mismatches. See, e.g., Hsu, et
al., 1994. Carcinogenesis 15: 1657-1662. According to an exemplary
embodiment, a probe based on a mutant EGFR sequence, e.g., a DEL-1
through DEL-5, G719S, G857V, L883S or L858R EGFR sequence, is hybridized
to a cDNA or other DNA product from a test cell(s). The duplex is
treated with a DNA mismatch repair enzyme, and the cleavage products,
if any, can be detected from electrophoresis protocols or the like.
See, e.g., U.S. Pat. No. 5,459,039.
In other embodiments, alterations in electrophoretic mobility will
be used to identify mutations in EGFR genes. For example, single
strand conformation polymorphism (SSCP) may be used to detect differences
in electrophoretic mobility between mutant and wild type nucleic
acids. See, e.g., Orita, et al., 1989. Proc. Natl. Acad. Sci. USA:
86: 2766; Cotton, 1993. Mutat. Res. 285: 125-144; Hayashi, 1992.
Genet. Anal. Tech. Appl. 9: 73-79. Single-stranded DNA fragments
of sample and control EGFR nucleic acids will be denatured and allowed
to renature. The secondary structure of single-stranded nucleic
acids varies according to sequence, the resulting alteration in
electrophoretic mobility enables the detection of even a single
base change. The DNA fragments may be labeled or detected with labeled
probes. The sensitivity of the assay may be enhanced by using RNA
(rather than DNA), in which the secondary structure is more sensitive
to a change in sequence. In one embodiment, the subject method utilizes
heteroduplex analysis to separate double stranded heteroduplex molecules
on the basis of changes in electrophoretic mobility. See, e.g.,
Keen, et al., 1991. Trends Genet. 7: 5.
In yet another embodiment, the movement of mutant or wild-type
fragments in polyacrylamide gels containing a gradient of denaturant
is assayed using denaturing gradient gel electrophoresis (DGGE).
See, e.g., Myers, et al., 1985. Nature 313: 495. When DGGE is used
as the method of analysis, DNA will be modified to insure that it
does not completely denature, for example by adding a GC clamp of
approximately 40 bp of high-melting GC-rich DNA by PCR. In a further
embodiment, a temperature gradient is used in place of a denaturing
gradient to identify differences in the mobility of control and
sample DNA. See, e.g., Rosenbaum and Reissner, 1987. Biophys. Chem.
265: 12753.
Examples of other techniques for detecting point mutations include,
but are not limited to, selective oligonucleotide hybridization,
selective amplification, or selective primer extension. For example,
oligonucleotide primers may be prepared in which the known mutation
is placed centrally and then hybridized to target DNA under conditions
that permit hybridization only if a perfect match is found. See,
e.g., Saiki, et al., 1986. Nature 324: 163; Saiki, et al., 1989.
Proc. Natl. Acad. Sci. USA 86: 6230. Such allele specific oligonucleotides
are hybridized to PCR amplified target DNA or a number of different
mutations when the oligonucleotides are attached to the hybridizing
membrane and hybridized with labeled target DNA.
Alternatively, allele specific amplification technology that depends
on selective PCR amplification may be used in conjunction with the
instant invention. Oligonucleotides used as primers for specific
amplification may carry the mutation of interest in the center of
the molecule (so that amplification depends on differential hybridization;
see, e.g., Gibbs, et al., 1989. Nucl. Acids Res. 17: 2437-2448)
or at the extreme 3'-terminus of one primer where, under appropriate
conditions, mismatch can prevent, or reduce polymerase extension
(see, e.g., Prossner, 1993. Tibtech. 11: 238). In addition it may
be desirable to introduce a novel restriction site in the region
of the mutation to create cleavage-based detection. See, e.g., Gasparini,
et al., 1992. Mol. Cell Probes 6: 1. It is anticipated that in certain
embodiments amplification may also be performed using Taq ligase
for amplification. See, e.g., Barany, 1991. Proc. Natl. Acad. Sci.
USA 88: 189. In such cases, ligation will occur only if there is
a perfect match at the 3'-terminus of the 5' sequence, making it
possible to detect the presence of a known mutation at a specific
site by looking for the presence or absence of amplification.
Solid Support and Probe
In an alternative embodiment, the detection of the presence or
absence of the at least one nucleic acid variance involves contacting
a nucleic acid sequence corresponding to the desired region of the
erbB1 gene, identified above, with a probe. The probe is able to
distinguish a particular form of the gene or the presence or a particular
variance or variances, e.g., by differential binding or hybridization.
Thus, exemplary probes include nucleic acid hybridization probes,
peptide nucleic acid probes, nucleotide-containing probes which
also contain at least one nucleotide analog, and antibodies, e.g.,
monoclonal antibodies, and other probes as discussed herein. Those
skilled in the art are familiar with the preparation of probes with
particular specificities. Those skilled in the art will recognize
that a variety of variables can be adjusted to optimize the discrimination
between two variant forms of a gene, including changes in salt concentration,
temperature, pH and addition of various compounds that affect the
differential affinity of GC vs. AT base pairs, such as tetramethyl
ammonium chloride. (See Current Protocols in Molecular Biology by
F. M. Ausubel, R. Brent, R. E. Kingston, D. D. Moore, J. G. Seidman,
K. Struhl and V. B. Chanda (Editors), John Wiley & Sons.)
Thus, in preferred embodiments, the detection of the presence or
absence of the at least one variance involves contacting a nucleic
acid sequence which includes at least one variance site with a probe,
preferably a nucleic acid probe, where the probe preferentially
hybridizes with a form of the nucleic acid sequence containing a
complementary base at the variance site as compared to hybridization
to a form of the nucleic acid sequence having a non-complementary
base at the variance site, where the hybridization is carried out
under selective hybridization conditions. Such a nucleic acid hybridization
probe may span two or more variance sites. Unless otherwise specified,
a nucleic acid probe can include one or more nucleic acid analogs,
labels or other substituents or moieties so long as the base-pairing
function is retained.
The probe may be designed to bind to, for example, at least three
continuous nucleotides on both sides of the deleted region of SEQ
ID NO: 495, SEQ ID NO: 497, or SEQ ID NO: 499. Such probes, when
hybridized under the appropriate conditions, will bind to the variant
form of EGFR, but will not bind to the wildtype EGFR.
Such hybridization probes are well known in the art (see, e.g.,
Sambrook et al., Eds., (most recent edition), Molecular Cloning:
A Laboratory Manual, (third edition, 2001), Vol. 1-3, Cold Spring
Harbor Laboratory, Cold Spring Harbor, N.Y.). Stringent hybridization
conditions will typically include salt concentrations of less than
about 1M, more usually less than about 500 mM and preferably less
than about 200 mM. Hybridization temperatures can be as low as 5.degree.
C., but are typically greater than 22.degree. C., more typically
greater than about 30.degree. C., and preferably in excess of about
37.degree. C. Longer fragments may require higher hybridization
temperatures for specific hybridization. Other factors may affect
the stringency of hybridization, including base composition and
length of the complementary strands, presence of organic solvents
and extent of base mismatching; the combination of parameters used
is more important than the absolute measure of any one alone. Other
hybridization conditions which may be controlled include buffer
type and concentration, solution pH, presence and concentration
of blocking reagents (e.g., repeat sequences, Cotl DNA, blocking
protein solutions) to decrease background binding, detergent type(s)
and concentrations, molecules such as polymers which increase the
relative concentration of the polynucleotides, metal ion(s) and
their concentration(s), chelator(s) and their concentrations, and
other conditions known or discoverable in the art. Formulas may
be used to predict the optimal melting temperature for a perfectly
complementary sequence for a given probe, but true melting temperatures
for a probe under a set of hybridization conditions must be determined
empirically. Also, a probe may be tested against its exact complement
to determine a precise melting temperature under a given set of
condition as described in Sambrook et al, "Molecular Cloning,"
3.sup.nd edition, Cold Spring Harbor Laboratory Press, 2001. Hybridization
temperatures can be systematically altered for a given hybridization
solution using a support associated with target polynucleotides
until a temperature range is identified which permits detection
of binding of a detectable probe at the level of stringency desired,
either at high stringency where only target polynucleotides with
a high degree of complementarity hybridize, or at lower stringency
where additional target polynucleotides having regions of complementarity
with the probe detectably hybridize above the background level provided
from nonspecific binding to noncomplementary target polynucleotides
and to the support. When hybridization is performed with potential
target polynucleotides on a support under a given set of conditions,
the support is then washed under increasing conditions of stringency
(typically lowered salt concentration and/or increased temperature,
but other conditions may be altered) until background binding is
lowered to the point where distinct positive signals may be seen.
This can be monitored in progress using a Geiger counter where the
probe is radiolabeled, radiographically, using a fluorescent imager,
or by other means of detecting probe binding. The support is not
allowed to dry during such procedures, or the probe may become irreversibly
bound even to background locations. Where a probe produces undesirable
background or false positives, blocking reagents are employed, or
different regions of the probe or different probes are used until
positive signals can be distinguished from background. Once conditions
are found that provide satisfactory signal above background, the
target polynucleotides providing a positive signal are isolated
and further characterized. The isolated polynucleotides can be sequenced;
the sequence can be compared to databank entries or known sequences;
where necessary, full-length clones can be obtained by techniques
known in the art; and the polynucleotides can be expressed using
suitable vectors and hosts to determine if the polynucleotide identified
encodes a protein having similar activity to that from which the
probe polynucleotide was derived. The probes can be from 10-50 nucleotides.
However, musch oarger probes can also be employed, e.g., 50-500
nucleotides or larger.
Solid Phase Support
The solid phase support of the present invention can be of any
solid materials and structures suitable for supporting nucleotide
hybridization and synthesis. Preferably, the solid phase support
comprises at least one substantially rigid surface on which oligonucleotides
or oligonucleotide primers can be immobilized. The solid phase support
can be made of, for example, glass, synthetic polymer, plastic,
hard non-mesh nylon or ceramic. Other suitable solid support materials
are known and readily available to those of skill in the art. The
size of the solid support can be any of the standard microarray
sizes, useful for DNA microarray technology, and the size may be
tailored to fit the particular machine being used to conduct a reaction
of the invention. Methods and materials for derivatization of solid
phase supports for the purpose of immobilizing oligonucleotides
are known to those skill in the art and described in, for example,
U.S. Pat. No. 5,919,523, the disclosure of which is incorporated
herein by reference.
The solid support can be provided in or be part of a fluid containing
vessel. For example, the solid support can be placed in a chamber
with sides that create a seal along the edge of the solid support
so as to contain the polymerase chain reaction (PCR) on the support.
In a specific example the chamber can have walls on each side of
a rectangular support to ensure that the PCR mixture remains on
the support and also to make the entire surface useful for providing
the primers.
The oligonucleotide or oligonucleotide primers of the invention
are affixed, immobilized, provided, and/or applied to the surface
of the solid support using any available means to fix, immobilize,
provide and/or apply the oligonucleotides at a particular location
on the solid support. For example, photolithography (Affymetrix,
Santa Clara, Calif.) can be used to apply the oligonucleotide primers
at particular position on a chip or solid support, as described
in the U.S. Pat. Nos. 5,919,523, 5,837,832, 5,831,070, and 5,770,722,
which are incorporated herein by reference. The oligonucleotide
primers may also be applied to a solid support as described in Brown
and Shalon, U.S. Pat. No. 5,807,522 (1998). Additionally, the primers
may be applied to a solid support using a robotic system, such as
one manufactured by Genetic MicroSystems (Woburn, Mass.), GeneMachines
(San Carlos, Calif.) or Cartesian Technologies (Irvine, Calif.).
In one aspect of the invention, solid phase amplification of target
polynucleotides from a biological sample is performed, wherein multiple
groups of oligonucleotide primers are immobilized on a solid phase
support. In a preferred embodiment, the primers within a group comprises
at least a first set of primers that are identical in sequence and
are complementary to a defined sequence of the target polynucleotide,
capable of hybridizing to the target polynucleotide under appropriate
conditions, and suitable as initial primers for nucleic acid synthesis
(i.e., chain elongation or extension). Selected primers covering
a particular region of the reference sequence are immobilized, as
a group, onto a solid support at a discrete location. Preferably,
the distance between groups is greater than the resolution of detection
means to be used for detecting the amplified products. In a preferred
embodiment, the primers are immobilized to form a microarray or
chip that can be processed and analyzed via automated, processing.
The immobilized primers are used for solid phase amplification of
target polynucleotides under conditions suitable for a nucleic acid
amplification means. In this manner, the presence or absence of
a variety of potential variances in the kinase domain of the erbB1
gene can be determined in one assay.
A population of target polynucleotides isolated from a healthy
individual can used as a control in determining whether a biological
source has at least one kinase activity increasing variance in the
kinase domain of the erb1 gene. Alternatively, target polynucleotides
isolated from healthy tissue of the same individual may be used
as a control as above.
An in situ-type PCR reactions on the microarrays can be conducted
essentially as described in e.g. Embretson et al, Nature 362:359-362
(1993); Gosden et al, BioTechniques 15(1):78-80 (1993); Heniford
et al Nuc. Acid Res. 21(14):3159-3166 (1993); Long et al, Histochemistry
99:151-162 (1993); Nuovo et al, PCR Methods and Applications 2(4):305-312
(1993); Patterson et al Science 260:976-979 (1993).
Alternatively, variances in the kinase domain of erbB1 can be determined
by solid phase techniques without performing PCR on the support.
A plurality of oligonucleotide probes, each containing a distinct
variance in the kinase domain of erbB1, in duplicate, triplicate
or quadruplicate, may be bound to the solid phase support. The presence
or absence of variances in the test biological sample may be detected
by selective hybridization techniques, known to those of skill in
the art and described above.
Mass Spectrometry
In another embodiment, the presence or absence of kinase activity
increasing nucleic acid variances in the kinase domain of the erbB1
gene are determined using mass spectrometry. To obtain an appropriate
quantity of nucleic acid molecules on which to perform mass spectrometry,
amplification may be necessary. Examples of appropriate amplification
procedures for use in the invention include: cloning (Sambrook et
al., Molecular Cloning: A Laboratory Manual, 3.sup.rd Edition, Cold
Spring Harbor Laboratory Press, 2001), polymerase chain reaction
(PCR) (C. R. Newton and A. Graham, PCR, BIOS Publishers, 1994),
ligase chain reaction (LCR) (Wiedmann, M., et al., (1994) PCR Methods
Appl. Vol. 3, Pp. 57-64; F. Barnay Proc. Natl. Acad. Sci USA 88,
189-93 (1991), strand displacement amplification (SDA) (G. Terrance
Walker et al., Nucleic Acids Res. 22, 2670-77 (1994)) and variations
such as RT-PCR (Higuchi, et al., Bio/Technology 11:1026-1030 (1993)),
allele-specific amplification (ASA) and transcription based processes.
To facilitate mass spectrometric analysis, a nucleic acid molecule
containing a nucleic acid sequence to be detected can be immobilized
to a solid support. Examples of appropriate solid supports include
beads (e.g. silica gel, controlled pore glass, magnetic, Sephadex/Sepharose,
cellulose), flat surfaces or chips (e.g. glass fiber filters, glass
surfaces, metal surface (steel, gold, silver, aluminum, copper and
silicon), capillaries, plastic (e.g. polyethylene, polypropylene,
polyamide, polyvinylidenedifluoride membranes or microtiter plates));
or pins or combs made from similar materials comprising beads or
flat surfaces or beads placed into pits in flat surfaces such as
wafers (e.g. silicon wafers).
Immobilization can be accomplished, for example, based on hybridization
between a capture nucleic acid sequence, which has already been
immobilized to the support and a complementary nucleic acid sequence,
which is also contained within the nucleic acid molecule containing
the nucleic acid sequence to be detected. So that hybridization
between the complementary nucleic acid molecules is not hindered
by the support, the capture nucleic acid can include a spacer region
of at least about five nucleotides in length between the solid support
and the capture nucleic acid sequence. The duplex formed will be
cleaved under the influence of the laser pulse and desorption can
be initiated. The solid support-bound base sequence can be presented
through natural oligoribo- or oligodeoxyribonucleotide as well as
analogs (e.g. thio-modified phosphodiester or phosphotriester backbone)
or employing oligonucleotide mimetics such as PNA analogs (see e.g.
Nielsen et al., Science, 254, 1497 (1991)) which render the base
sequence less susceptible to enzymatic degradation and hence increases
overall stability of the solid support-bound capture base sequence.
Prior to mass spectrometric analysis, it may be useful to "condition"
nucleic acid molecules, for example to decrease the laser energy
required for volatilization and/or to minimize fragmentation. Conditioning
is preferably performed while a target detection site is immobilized.
An example of conditioning is modification of the phosphodiester
backbone of the nucleic acid molecule (e.g. cation exchange), which
can be useful for eliminating peak broadening due to a heterogeneity
in the cations bound per nucleotide unit. Contacting a nucleic acid
molecule with an alkylating agent such as alkyliodide, iodoacetamide,
.beta.-iodoethanol, 2,3-epoxy-1-propanol, the monothio phosphodiester
bonds of a nucleic acid molecule can be transformed into a phosphotriester
bond. Likewise, phosphodiester bonds may be transformed to uncharged
derivatives employing trialkylsilyl chlorides. Further conditioning
involves incorporating nucleotides which reduce sensitivity for
depurination (fragmentation during MS) such as N7- or N9-deazapurine
nucleotides, or RNA building blocks or using oligonucleotide triesters
or incorporating phosphorothioate functions which are alkylated
or employing oligonucleotide mimetics such as PNA.
For certain applications, it may be useful to simultaneously detect
more than one (mutated) loci on a particular captured nucleic acid
fragment (on one spot of an array) or it may be useful to perform
parallel processing by using oligonucleotide or oligonucleotide
mimetic arrays on various solid supports. "Multiplexing"
can be achieved by several different methodologies. For example,
several mutations can be simultaneously detected on one target sequence
by employing corresponding detector (probe) molecules (e.g. oligonucleotides
or oligonucleotide mimetics). However, the molecular weight differences
between the detector oligonucleotides D1, D2 and D3 must be large
enough so that simultaneous detection (multiplexing) is possible.
This can be achieved either by the sequence itself (composition
or length) or by the introduction of mass-modifying functionalities
M1-M3 into the detector oligonucleotide.
Preferred mass spectrometer formats for use in the invention are
matrix assisted laser desorption ionization (MALDI), electrospray
(ES), ion cyclotron resonance (ICR) and Fourier Transform. Methods
of performing mass spectrometry are known to those of skill in the
art and are further described in Methods of Enzymology, Vol. 193:
"Mass Spectrometry" (J. A. McCloskey, editor), 1990, Academic
Press, New York.
Sequencing
In other preferred embodiments, determining the presence or absence
of the at least one kinase activity increasing nucleic acid variance
involves sequencing at least one nucleic acid sequence. The sequencing
involves the sequencing of a portion or portions of the kinase domain
of erbB1 which includes at least one variance site, and may include
a plurality of such sites. Preferably, the portion is 500 nucleotides
or less in length, more preferably 100 nucleotides or less, and
most preferably 45 nucleotides or less in length. Such sequencing
can be carried out by various methods recognized by those skilled
in the art, including use of dideoxy termination methods (e.g.,
using dye-labeled dideoxy nucleotides), minisequencing, and the
use of mass spectrometric methods.
Immunodetection
In one embodiment, determining the presence or absence of the at
least one kinase activity increasing nucleic acid variance involves
determining the activation state of downstream targets of EGFR.
The inventors of the present application have compared the phosphorylation
status of the major downstream targets of EGFR. For example, the
EGF-induced activation of Erk1 and Erk2, via Ras, of Akt via PLC.gamma./PI3K,
and of STAT3 and STAT5 via JAK2, has been examined. Erk1 and Erk2,
via Ras, Akt via PLC.gamma./PI3K, and STAT3 and STAT5 via JAK2 are
essential downstream pathways mediating oncogenic effects of EGFR(R.
N. Jorissen et al., Exp. Cell Res. 284, 31 (2003)).
The inventors of the present application have shown that EGF-induced
Erk activation is indistinguishable among cells expressing wild-type
EGFR or either of the two activating EGFR mutants.
In contrast, phosphorylation of both Akt and STAT5 was substantially
elevated in cells expressing either of the mutant EGFRs. Increased
phosphorylation of STAT3 was similarly observed in cells expressing
mutant EGFRs. Thus, the selective EGF-induced autophosphorylation
of C-terminal tyrosine residues within EGFR mutants is well correlated
with the selective activation of downstream signaling pathways.
In one embodiment of the present application, the presence of EGFR
mutations can be determined using immunological techniques well
known in the art, e.g., antibody techniques such as immunohistochemistry,
immunocytochemistry, FACS scanning, immunoblotting, radioimmunoassays,
western blotting, immunoprecipitation, enzyme-linked immunosorbant
assays (ELISA), and derivative techniques that make use of antibodies
directed against activated downstream targets of EGFR. Examples
of such targets include, for example, phosphorylated STAT3, phosphorylated
STAT5, and phosphorylated Akt. Using phospho-specific antibodies,
the activation status of STAT3, STAT5, and Akt can be determined.
Activation of STAT3, STAT5, and Akt are useful as a diagnostic indicator
of activating EGFR mutations.
In one embodiment of the present invention, the presence of activated
(phosphorylated) STAT5, STAT3, or Akt indicates that an EGFR targeting
treatment is likely to be effective.
The invention provides a method of screening for variants in the
kinase domain of the erbB1 gene in a test biological sample by immunohistochemical
or immunocytochemical methods.
Immunohistochemistry ("IHC") and immunocytochemistry
("ICC") techniques, for example, may be used. IHC is the
application of immunochemistry to tissue sections, whereas ICC is
the application of immunochemistry to cells or tissue imprints after
they have undergone specific cytological preparations such as, for
example, liquid-based preparations. Immunochemistry is a family
of techniques based on the use of a specific antibody, wherein antibodies
are used to specifically target molecules inside or on the surface
of cells. The antibody typically contains a marker that will undergo
a biochemical reaction, and thereby experience a change color, upon
encountering the targeted molecules. In some instances, signal amplification
may be integrated into the particular protocol, wherein a secondary
antibody, that includes the marker stain, follows the application
of a primary specific antibody.
Immunoshistochemical assays are known to those of skill in the
art (e.g., see Jalkanen, et al., J. Cell. Biol. 101:976-985 (1985);
Jalkanen, et al., J. Cell. Biol. 105:3087-3096 (1987).
Antibodies, polyclonal or monoclonal, can be purchased from a variety
of commercial suppliers, or may be manufactured using well-known
methods, e.g., as described in Harlow et al., Antibodies: A Laboratory
Manual, 2nd Ed; Cold Spring Harbor Laboratory Press, Cold Spring
Harbor, N.Y. (1988). In general, examples of antibodies useful in
the present invention include anti-phospho-STAT3, anti-phospho-STAT5,
and anti-phospho-Akt antibodies. Such antibodies can be purchased,
for example, from Upstate Biotechnology (Lake Placid, N.Y.), New
England Biolabs (Beverly, Mass.), NeoMarkers (Fremont, Calif.)
Typically, for immunohistochemistry, tissue sections are obtained
from a patient and fixed by a suitable fixing agent such as alcohol,
acetone, and paraformaldehyde, to which is reacted an antibody.
Conventional methods for immunohistochemistry are described in Harlow
and Lane (eds) (1988) In "Antibodies A Laboratory Manual",
Cold Spring Harbor Press, Cold Spring Harbor, N.Y.; Ausbel et al
(eds) (1987), in Current Protocols In Molecular Biology, John Wiley
and Sons (New York, N.Y.). Biological samples appropriate for such
detection assays include, but are not limited to, cells, tissue
biopsy, whole blood, plasma, serum, sputum, cerebrospinal fluid,
breast aspirates, pleural fluid, urine and the like.
For direct labeling techniques, a labeled antibody is utilized.
For indirect labeling techniques, the sample is further reacted
with a labeled substance.
Alternatively, immunocytochemistry may be utilized. In general,
cells are obtained from a patient and fixed by a suitable fixing
agent such as alcohol, acetone, and paraformaldehyde, to which is
reacted an antibody. Methods of immunocytological staining of human
samples is known to those of skill in the art and described, for
example, in Brauer et al., 2001 (FASEB J, 15, 2689-2701), Smith-Swintosky
et al., 1997.
Immunological methods of the present invention are advantageous
because they require only small quantities of biological material.
Such methods may be done at the cellular level and thereby necessitate
a minimum of one cell. Preferably, several cells are obtained from
a patient affected with or at risk for developing cancer and assayed
according to the methods of the present invention.
Other Diagnostic Methods
An agent for detecting mutant EGFR protein is an antibody capable
of binding to mutant EGFR protein, preferably an antibody with a
detectable label. Antibodies can be polyclonal, or more preferably,
monoclonal. An intact antibody, or a fragment thereof (e.g., F.sub.ab
or F.sub.(ab)2) can be used. The term "labeled", with
regard to the probe or antibody, is intended to encompass direct
labeling of the probe or antibody by coupling (i.e., physically
linking) a detectable substance to the probe or antibody, as well
as indirect labeling of the probe or antibody by reactivity with
another reagent that is directly labeled. Examples of indirect labeling
include detection of a primary antibody using a fluorescently-labeled
secondary antibody and end-labeling of a DNA probe with biotin such
that it can be detected with fluorescently-labeled streptavidin.
The term "biological sample" is intended to include tissues,
cells and biological fluids isolated from a subject, as well as
tissues, cells and fluids present within a subject. That is, the
detection method of the invention can be used to detect mutant EGFR
mRNA, protein, or genomic DNA in a biological sample in vitro as
well as in vivo. For example, in vitro techniques for detection
of mutant EGFR mRNA include Northern hybridizations and in situ
hybridizations. In vitro techniques for detection of mutant EGFR
protein include enzyme linked immunosorbent assays (ELISAs), Western
blots, immunoprecipitations, and immunofluorescence. In vitro techniques
for detection of mutant EGFR genomic DNA include Southern hybridizations.
Furthermore, in vivo techniques for detection of mutant EGFR protein
include introducing into a subject a labeled anti-mutant EGFR protein
antibody. For example, the antibody can be labeled with a radioactive
marker whose presence and location in a subject can be detected
by standard imaging techniques.
In one embodiment, the biological sample contains protein molecules
from the test subject. Alternatively, the biological sample can
contain mRNA molecules from the test subject or genomic DNA molecules
from the test subject.
In another embodiment, the methods further involve obtaining a
control biological sample from a control subject, contacting the
control sample with a compound or agent capable of detecting mutant
EGFR protein, mRNA, or genomic DNA, such that the presence of mutant
EGFR protein, mRNA or genomic DNA is detected in the biological
sample, and comparing the presence of mutant EGFR protein, mRNA
or genomic DNA in the control sample with the presence of mutant
EGFR protein, mRNA or genomic DNA in the test sample.
In a different embodiment, the diagnostic assay is for mutant EGFR
activity. In a specific embodiment, the mutant EGFR activity is
a tyrosine kinase activity. One such diagnostic assay is for detecting
EGFR-mediated phosphorylation of at least one EGFR substrate. Levels
of EGFR activity can be assayed for, e.g., various mutant EGFR polypeptides,
various tissues containing mutant EGFR, biopsies from cancer tissues
suspected of having at least one mutant EGFR, and the like. Comparisons
of the levels of EGFR activity in these various cells, tissues,
or extracts of the same, can optionally be made. In one embodiment,
high levels of EGFR activity in cancerous tissue is diagnostic for
cancers that may be susceptible to treatments with one or more tyrosine
kinase inhibitor. In related embodiments, EGFR activity levels can
be determined between treated and untreated biopsy samples, cell
lines, transgenic animals, or extracts from any of these, to determine
the effect of a given treatment on mutant EGFR activity as compared
to an untreated control.
Method of Treating a Patient
In one embodiment, the invention provides a method for selecting
a treatment for a patient affected by or at risk for developing
cancer by determining the presence or absence of at least one kinase
activity increasing nucleic acid variance in the kinase domain of
the erbB1 gene. In another embodiment, the variance is a plurality
of variances, whereby a plurality may include variances from one,
two, three or more gene loci.
In certain embodiments, the presence of the at least one variance
is indicative that the treatment will be effective or otherwise
beneficial (or more likely to be beneficial) in the patient. Stating
that the treatment will be effective means that the probability
of beneficial therapeutic effect is greater than in a person not
having the appropriate presence of the particular kinase activity
increasing nucleic acid variance(s) in the kinase domain of the
erbB1 gene.
The treatment will involve the administration of a tyrosine kinase
inhibitor. The treatment may involve a combination of treatments,
including, but not limited to a tyrosine kinase inhibitor in combination
with other tyrosine kinase inhibitors, chemotherapy, radiation,
etc.
Thus, in connection with the administration of a tyrosine kinase
inhibitor, a drug which is "effective against" a cancer
indicates that administration in a clinically appropriate manner
results in a beneficial effect for at least a statistically significant
fraction of patients, such as a improvement of symptoms, a cure,
a reduction in disease load, reduction in tumor mass or cell numbers,
extension of life, improvement in quality of life, or other effect
generally recognized as positive by medical doctors familiar with
treating the particular type of disease or condition.
In a preferred embodiment, the compound is an anilinoquinazoline
or synthetic anilinoquinazoline. European. Patent Publication No.
0566226 discloses anilinoquinazolines which have activity against
epidermal growth factor (EGF) receptor tyrosine kinase. It is also
known from European Patent Applications Nos. 0520722 and 0566226
that certain 4-anilinoquinazoline derivatives are useful as inhibitors
of receptor tyrosine kinases. The very tight structure-activity
relationships shown by these compounds suggests a clearly-defined
binding mode, where the quinazoline ring binds in the adenine pocket
and the anilino ring binds in an adjacent, unique lipophilic pocket.
Three 4-anilinoquinazoline analogues (two reversible and one irreversible
inhibitor) have been evaluated clinically as anticancer drugs. Denny,
Farmaco January-February 2001; 56(1-2):51-6. Alternatively, the
compound is EKB-569, an inhibitor of EGF receptor kinase (Torrance
et al., Nature Medicine, vol. 6, No. 9, September 2000, p. 1024).
In a most preferred embodiment, the compound is gefitinib (IRESSA.RTM.)
or erlotinib (TARCEVA.RTM.).
Treatment targeting cancer cells containing at least one mutant
EGFR described herein may be administered alone or in combination
with any other appropriate anti-cancer treatment and/or therapeutic
agent known to one skilled in the art. In one embodiment, treatment
of a pathology, such as a cancer, is provided comprising administering
to a subject in need thereof therapeutically effective amounts of
a compound that inhibits EGFR kinase activity, such as gefitinib,
erlotinib, etc., administered alone or in combination with at least
one other anti-cancer agent or therapy. Inhibition of activated
protein kinases through the use of targeted small molecule drugs
or antibody-based strategies has emerged as an effective approach
to cancer therapy. See, e.g., G. D. Demetri et al., N. Engl. J.
Med. 347, 472 (2002); B. J. Druker et al., N. Engl. J. Med. 344,
1038 (2001); D. J. Slamon et al., N. Engl. J. Med. 344, 783 (2001).
In one embodiment, the anti-cancer agent is at least one chemotherapeutic
agent. In a related embodiment, the anti-cancer agent is at least
one radiotherapy. In a variant embodiment, the anti-cancer therapy
is an antiangiogenic therapy (e.g., endostatin, angiostatin, TNP-470,
Caplostatin (Stachi-Fainaro et al., Cancer Cell 7(3), 251 (2005))
The therapeutic agents may be the same or different, and may be,
for example, therapeutic radionuclides, drugs, hormones, hormone
antagonists, receptor antagonists, enzymes or proenzymes activated
by another agent, autocrines, cytokines or any suitable anti-cancer
agent known to those skilled in the art. In one embodiment, the
anti-cancer agent is Avastin, an anti-VEGF antibody proven successful
in anti-angiogenic therapy of cancer against both solid cancers
and hematological malignancies. See, e.g., Ribatti et al. 2003 J
Hematother Stem Cell Res. 12(1), 11-22. Toxins also can be used
in the methods of the present invention. Other therapeutic agents
useful in the present invention include anti-DNA, anti-RNA, radiolabeled
oligonucleotides, such as antisense oligonucleotides, anti-protein
and anti-chromatin cytotoxic or antimicrobial agents. Other therapeutic
agents are known to those skilled in the art, and the use of such
other therapeutic agents in accordance with the present invention
is specifically contemplated.
The antitumor agent may be one of numerous chemotherapy agents
such as an alkylating agent, an antimetabolite, a hormonal agent,
an antibiotic, an antibody, an anti-cancer biological, gleevec,
colchicine, a vinca alkaloid, L-asparaginase, procarbazine, hydroxyurea,
mitotane, nitrosoureas or an imidazole carboxamide. Suitable agents
are those agents that promote depolarization of tubulin or prohibit
tumor cell proliferation. Chemotherapeutic agents contemplated as
within the scope of the invention include, but are not limited to,
anti-cancer agents listed in the Orange Book of Approved Drug Products
With Therapeutic Equivalence Evaluations, as compiled by the Food
and Drug Administration and the U.S. Department of Health and Human
Services. Nonlimiting examples of chemotherapeutic agents include,
e.g., carboplatin and paclitaxel. Treatments targeting EGFR kinase
activity can also be administered together with radiation therapy
treatment. Additional anti-cancer treatments known in the art are
contemplated as being within the scope of the invention.
The therapeutic agent may be a chemotherapeutic agent. Chemotherapeutic
agents are known in the art and include at least the taxanes, nitrogen
mustards, ethylenimine derivatives, alkyl sulfonates, nitrosoureas,
triazenes; folic acid analogs, pyrimidine analogs, purine analogs,
vinca alkaloids, antibiotics, enzymes, platinum coordination complexes,
substituted urea, methyl hydrazine derivatives, adrenocortical suppressants,
or antagonists. More specifically, the chemotherapeutic agents may
be one or more agents chosen from the non-limiting group of steroids,
progestins, estrogens, antiestrogens, or androgens. Even more specifically,
the chemotherapy agents may be azaribine, bleomycin, bryostatin-1,
busulfan, carmustine, chlorambucil, carboplatin, cisplatin, CPT-11,
cyclophosphamide, cytarabine, dacarbazine, dactinomycin, daunorubicin,
dexamethasone, diethylstilbestrol, doxorubicin, ethinyl estradiol,
etoposide, fluorouracil, fluoxymesterone, gemcitabine, hydroxyprogesterone
caproate, hydroxyurea, L-asparaginase, leucovorin, lomustine, mechlorethamine,
|