Note: Descriptions are shown in the official language in which they were submitted.
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METHOD AND COMPOSITIONS FOR THE DIAGNOSIS AND
TREATMENT OF NON-SMALL CELL LUNG CANCER USING GENE
EXPRESSION PROFILES
This application is a division of Canadian Patent Application Serial
No. 2,480,045. The claims of the present application are generally directed
to methods that identify the expression. profile of at least one of the c-myc,
E2F-1 and p21 genes, which are associated with non-small cell lung cancer,
in both normal lung and diseased lung. These diagnostic markers are useful
in identifying disease states, disease progression and drug efficacy.
Accordingly, the retention of any objects or features which may be
more particularly related to the parent application or a separate divisional
thereof should not be regarded as rendering the teachings and claiming
ambiguous or inconsistent with the subject matter defined in the claims of
the divisional application presented herein when seeking to interpret the
scope thereof and the basis in this disclosure for the claims recited herein.
BACKGROUND OF THE INVENTION
Non-small cell lung cancer (NSCLC) is the most common type of
bronchogenic carcinoma. Although chemotherapeutic regimens with greater
efficacy continue to be developed, the best regimens presently give an
overall regression rate of only 30-50%. This lack of response is attributable
to resistance that is present de novo or develops in response to treatment. It
is believed that mechanisms of chemoresistance likely involve multiple gene
products. It is important to define the role of specific genes involved in
tumor
development and growth and to identify and quantify those genes and gene
products that can serve as targets for diagnosis, prevention, monitoring and
treatment of cancer.
In certain instances, therapeutic agents that are initially effective
become ineffective or less effective for a patient over time. The same
therapeutic agent can continue to be effective for a longer period of time for
a different patient. Further, the therapeutic agents can be ineffective or
harmful to still other patients. Therefore, it would be beneficial to identify
CA 02820020 2013-06-26
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genes and/or gene products that could serve as markers with respect to
cancers and to given therapeutic agents. The ability to make such
predictions and corrections in the treatment make it possible to more
accurately make decisions on the therapeutic regime at an earlier stage in
time in the course of a treatment of a patient.
Currently, cisplatin and carboplatin are among the most widely used
cytotoxic anticancer drugs. However, resistance to these drugs through de
novo or induced mechanisms undermines their curative potential. Perez,
R.P., Cellular and molecular determinants of cisplatin resistance, Eur. J.
Cancer (1998), 34, 1535-1542. Recently, understanding regarding potential
modes of chemoresistance to platinum compounds has been obtained
through studies correlating cytotoxicity with nucleotide excision-repair (NER)
(Dijt, F., Fitchinger-Schepman, A.M., Berends, F., Reedikj, J., Formation and
repair of cisplatin-induced adducts to DNA in cultured normal and repair-
deficient human fibroblasts, Cancer Res. (1988), 48, 6058-6062. Zamble,
D.B., Lippard, S.J., Cisplatin and DNA repair in cancer chemotherapy,
Trends Biochem Sci (1995), 20, 435-439. States, J.C., Reed, E., Enhanced
XPA mRNA levels in cisplatin-resistant human ovarian cancer are not
associated with XPA mutations or gene amplifications, Cancer Lett. (1996),
108, 233-237. Ferry, K.V., Fink, D., Johnson, S.W., Hamilton, T.C., Howell,
S.B., Quantitation of platinum-DNA adduct repair in mismatch repair
deficient and proficient human colorectal cancer cell lines using an in vitro
DNA repair assay, Proc. Am. Assoc. Cancer Res. (1997), abstract, 38, 359.
Jordan, P., Carmo-Fonseca, M., Molecular mechanisms involved in cisplatin
cytotoxicity, Cell Mol. Life Sci. (2000), 57, 1229-1235. Kartalou, M.,
Essingmann, J.M., Mechanisms of resistance to cisplatin, Mutat. Res. (2001
), 478, 23-43) or drug uptake/efflux (Kartalou, M., Essingmann, J.M.,
Mechanisms of resistance to cisplatin, Mutat. Res. (2001), 478, 23-43.
Berger, W., Elbling, L., Hauptmann, E., Micksche, M., Expression of the
multidrug resistance-associated protein (MRP) and chemoresistance of
human non-small-cell lung cancer cells, Int. J. Cancer (1997), 73, 84-93.
Borst, P., Kool, M., Evers, R., Do cMOAT (MRP2), other MRP homologues,
and LRP play a role in MDR? Cancer Biol. (1997), 8, 205-213. Young, LC,
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Campling, B.G., Voskoglou-Nomikos, T., Cole, S.P.C., Deeley, R.G.,
Gerlach, J.H., Expression of multidrug. resistance protein-related genes in
lung cancer: correlation with drug response, Clin. Cancer Res. (1999), 5,
673-680. Berger, W., Elbling, L, Micksche, M., Expression of the major vault
protein LRP in human non-small-cell lung cancer cells: activation by short-
term exposure to antineoplastic drugs, Int. J. Cancer (2000), 88, 293-300.
Borst, P., Evers, R., Kool, M., Wijnholds, J., A family of drug transporters:
the multidrug resistance-associated proteins, J Nat. Cancer Inst. (2000), 92,
1295-1302. Oguri, T., lsobe, T., Suzuki, T., Nishio, K., Fujiwara, Y., Katoh,
0., Yamakido, M., Increased expression of the MRP5 gene is associated
with exposure to platinum drugs in lung cancer, Int. J. Cancer (2000), 86,
95-100.
Current advances in technology, including microarrays and
quantitative RT-PCR methods, are allowing classification of cancer types on
the basis of functional genomics as opposed to histomorphology. Golub,
T.R., Slonim, D.K., Tamayo, P., Huard, 0, Gaasenbeek, M., Mesirov, J.P.,
Coller, H., Loh, M.L., Downing, J.R., Caligiuri, M.A., Bloomfield, CD.,
Lander, E.S., Molecular classification of cancer: class discovery and class
prediction by gene expression monitoring, Science (1999), 286, 531-537.
Alizadeh, A.A., Eisen, M.B., Davis, R.E., Ma, C, Lossos, I.S., Rosenwald, A.,
Boldrick, J.C., Sabet, H., Tran, T., Yu, X., Powell, J.I., Yang, L, Marti,
G.E.,
Moore, T., Hudson, Jr., J., Lu, L., Lewis, D.B., Tibshirani, R., Sherlock, G.,
Chan, W.0, Greiner, T.C., Weisenburger, D.D., Armitage, JØ, Warnke, R.,
Staudt, L.M., et al., Distinct types of diffuse large B-cell lymphoma
identified
by gene expression profiling, Nature (2000), 403, 503-511. For example,
they may allow for the discovery of predictive markers based on gene
expression profiles. Microarray screening analysis currently is being
investigated to predict chemotherapeutic sensitivity based on gene
expression profiles. Scherf, U., Ross, DI., Waltham, M., Smith, L.H., Lee,
J.K., Tanabe, L, Kohn, K.W., Reinhold, W.C., Myers, T.G., Andrews, D.T.,
Scudiero, D.A., Eisen, MB., Sausville, E.A., Pommier, Y., Botstein, D.,
Brown, P.O., Weinstein, J.N., A gene expression database for the molecular
pharmacology of cancer, Nat. Genet. (2000), 24, 236-244. Kihara, C.
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Tsunoda, T., Tanaka, T., Yamana, H., Furukawa, Y., Ono, K., Kitahara, 0.,
Zembutsu, H., Yanagawa, R., Hirata, K., Takagi, T., Nakamura, Y.,
Prediction of sensitivity of esophageal tumors to adjuvant chemotherapy by
cDNA microarray analysis of gene-expression profiles, Cancer Res. (2001),
61, 6474-6479. Zembutsu, H., Ohnishi, Y., Tsunoda, T., Furukawa, Y.,
Katagiri, T., Ueyama, Y., Tamaoki, N., Nomura, T., Kitahara, 0., Yanagawa,
R., Hirata, K., Nakamura, Y., Genome-wide cDNA microarray screening to
correlate gene expression profiles with sensitivity of 85 human cancer
xenografts to anticancer drugs, Cancer Res. (2002), 62, 518-527. An
advantage of microarray analysis is that thousands of genes may be
simultaneously evaluated. However, it is generally recognized that, due to
lack of standardization, relatively low sensitivity and relatively poor lower
thresholds of detection, microarray assessments need to be confirmed with
follow-up quantitative methods. StaRT-PCR is a method that allows for
rapid, reproducible, standardized, quantitative measurements for many
genes simultaneously. Willey, J.C, Crawford, E.L, Jackson, CM., Weaver,
D.A., Hoban, J.C, Khuder, S.A., DeMuth, J.P., Expression measurement of
many genes simultaneously by quantitative RT-PCR using standardized
mixtures of competitive templates, Am. J. Respir. Cell Mol. Biol. (1998), 19,
6-17. Weaver, et al. Comparison of expression patterns by microarray and
standardized RT-PCR analyses in lung cancer cell lines with varied
sensitivity to carboplatin. Proc. Am. Assoc. Cancer Res. 2001 (abstract) 42,
606.
StaRT-PCR can also be used to more accurately diagnose lung
cancer in small biopsy tissues. Warner, et al. "High c-myc x E2F-1/p21 may
augment cytologic diagnosis of NSCLC" Prod. Am. Assoc. Cancer Res. Vol.
43, abstract 3738, March 2002; Weaver, et al. Gene expression modeling of
cisplatin chemoresistance in non-small cell lung cancer cell lines utilizing
standardized RT StarRT-PCR" Prod. Am. Assoc. Cancer Res. Vol. 43,
abstract 5471, March 2002.
SUMMARY OF THE INVENTION
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The present invention identifies patterns of individual, interactive
gene expression and/or indices (IGEI) comprising the expression values of
multiple genes which, in one instance, are more effective markers of
chemoresistant non-small cell lung cancer (NSCLC) tumors than expression
5 values of individual genes, and in another instance, may be used to more
accurately diagnose lung cancer in small biopsy tissues.
The present invention is directed to the identification and use of
markers that can be used to determine the sensitivity of cancer cells to a
therapeutic agent. More specifically, the invention features "a number of
markers" that are variably expressed in cancer tissue and can be used to
determine the sensitivity of cancer cells to a therapeutic agent. Still more
specifically, the invention features "interactive gene expression indices"
(IGEI) useful for assessment of biological samples to prospectively identify
the usefulness of therapeutic agents.
The present invention thus provides gene expression profiles which
serve as useful diagnostic markers as well as markers that can be used to
monitor disease states, disease progression, drug toxicity, drug efficacy and
drug metabolism.
The present invention further provides a method to determine
whether an agent or combination of agents can be used to reduce the
growth of cancer cells as well as determining new agents for the treatment
of cancer
Various embodiments of the present invention are directed to uses of
the identified markers whose expression is correlated with accurate
diagnosis of lung cancer cells or tissue compared to normal tissues, and
other markers whose expression is correlated with sensitivity to treatment
with a therapeutic agent. In particular, the present invention provides,
without limitation: 1) methods for determining whether a particular tissue is
lung cancer or non cancer tissue; 2) methods for monitoring the
effectiveness of therapeutic agents used for the treatment of cancer; 3)
methods for developing new therapeutic agents for the treatment of cancer;
and 4) methods for identifying combinations of therapeutic agents for the
treatment of cancer.
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By examining and quantifying the expression of one or more of the
identified markers in a sample of cancer cells, it is further possible to
determine which therapeutic agent or combination of agents will be most
likely to reduce the growth rate of the cancer and can further be used in
selecting appropriate treatment agents.
By examining and quantifying the expression of one or more of the
identified markers in a sample of cancer cells, it is also possible to
determine which therapeutic agent or combination of agents will be the least
likely to reduce the growth rate of the cancer.
By examining and quantifying the expression of one or more of the
identified markers, it is also possible to eliminate inappropriate therapeutic
agents.
By examining and quantifying the expression of one or more
identified markers when cancer cells or a cancer cell line is exposed to a
potential anti-cancer agent, it is possible to identify the efficacy of new
anti-
cancer agents.
Further, by examining and quantifying the expression of one or more
of the identified markers in a sample of cancer cells taken from a patient
during the course of therapeutic treatment, it is possible to determine
whether the therapeutic treatment is continuing to be effective or whether
the cancer has become resistant (refractory) to the therapeutic treatment.
These determinations can be made on a patient-by-patient basis or on an
agent by agent (or combination of agents) basis. It may also be possible to
determine whether or not a particular therapeutic treatment is likely to
benefit a particular patient or group/class of patients, or whether a
particular
treatment should be continued.
The present invention further provides previously unknown or
unrecognized targets for the development of anti-cancer agents, such as
chemotherapeutic compounds.
The identified interactive gene expression indices (IGE!) of the
present invention are useful as targets in developing treatments (either for a
single agent or for multiple agents) for cancer.
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The present invention identifies the global changes in gene
expression associated with lung cancer by examining gene expression in
tissue from normal lung. The present invention also identifies expression
profiles which serve as useful diagnostic markers as well as markers that
can be used to monitor disease states,, disease progression, drug toxicity,
drug efficacy and drug metabolism.
In some preferred embodiments, the methods, genes, and IGEI
described herein are useful to identify cisplatin resistant cancers (in
contrast
to diagnosing cancers from normal tissues). Such embodiments may include
detecting the expression level of one or more genes selected from a group
consisting of ERCC2, ABCC5, XPA and XRCC1.
In some preferred embodiments, the method may include detecting
the expression level of one or more genes selected from a group consisting
of ERCC2/XPC, ABCC5/GTF2H2, ERCC2/GTF2H2, XPA/XPC and
XRCC 1/XPC
In some preferred embodiments, the method may include detecting
the expression level of one or more genes selected from a group consisting
of ABCC5/GTF2H2, and ERCC2/GTF2H2.
The invention also includes methods of detecting the progression of
NSCLC and/or differentiating small cell lung cancer (SCLC) and/or
nonmetastatic from metastatic disease. For instance, methods of the
invention include detecting the progression of NSCLC in a patient
comprising the step of detecting the level of expression in a tissue sample of
two or more genes from Tables 1 and/or 5; wherein differential expression of
the genes in Tables 1 and/or 5 is indicative of NSCLC progression. In some
preferred embodiments, one or more genes may be selected from a group
consisting of the genes listed in Table 5.
In some aspects, the present invention provides a method of
monitoring the treatment of a patient with NSCLC, comprising administering
a pharmaceutical composition to the patient and preparing a gene
expression profile from a cell or tissue sample from the patient and
comparing the patient gene expression profile to a gene expression from a
cell population comprising normal lung cells or to a gene expression profile
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from a cell population comprising lung cancer cells or to both. In some
preferred embodiments, the gene profile will include the expression level of
one or more genes in Tables 1 and 5. in other preferred embodiments, one
or more genes may be selected from a group consisting of the genes listed
in Table 5.
In another aspect, the present invention provides a method of treating
a patient with NSCLC, comprising administering to the patient a
pharmaceutical composition, wherein the composition alters the expression
of at least one gene in Tables 1 and 5, preparing a gene expression profile
from a cell or tissue sample from the patient comprising tumor cells and
comparing the patient expression profile to a gene expression profile from
an untreated cell population comprising NSCLC cells. In some preferred
embodiments, one or more genes may be selected from a group consisting
of the genes listed in Table 5.
The invention includes methods of diagnosing the presence or
absence of lung cancer in a patient comprising the step of detecting the
level of expression in a tissue sample of an IGEI comprising c-myc x E2F-
1/p21 (Sequence ID Nos. 40-48 since each gene has 3 primer sequences)
in which the c-myc gene expression value (molecules/10613-actin molecules)
is multiplied times the E2F-1 expression value and this product is divided by
the p21 gene expression value.
The c-myc x E2F-1/p21 index may also be used as a marker for the
monitoring of disease progression, for instance, the development of lung
cancer. For instance, a lung tissue sample or other sample from a patient
may be assayed by any of the methods described herein, and the
expression levels in the sample of c-myc x E2F-1/p21 may be compared to
the expression levels found in normal lung tissue, tissue from SCLC,
metastatic lung cancer or NSCLC tissue. Comparison of the expression
data, as well as available sequence or other information may be done by
researcher or diagnostician or may be done with the aid of a computer and
databases as described herein.
The invention further includes methods of screening for an agent
capable of modulating the onset or progression of NSCLC, comprising the
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steps of exposing a cell to the agent; and detecting the expression level of
the c-myc x E2F-1/p21 index.
According to one aspect of the present invention, the genes identified
in Tables 1 and 5 may be used as markers to evaluate the effects of a
candidate drug or agent on a cell or tissue sample, for instance, a lung
cancer cell or tissue sample. A candidate drug or agent can be screened for
the ability to simulate the transcription or expression of a given marker or
set
of marker genes (drug targets) or to down-regulate or counteract the
transcription or expression of a marker or markers. According to the present
io invention, one
can also compare the specificity of drugs' effects on gene
expression markers and comparing them. More specific drugs may have
fewer transcriptional targets. Similar sets of markers identified for two
drugs
indicate a similarity of effects.
Any of the methods of the invention described above may include the
detection and quantification of at least 2 genes from the Tables 1 and/or 5 or
c-myc x E2F-1/p21. Preferred methods may detect and quantify all or nearly
all of the genes in the tables. In some preferred embodiments, one or more
genes may be selected from a group consisting of the genes listed in Table
5.
According to another aspect, the present invention relates to a
method of diagnosing non small cell lung cancer in a patient, comprising: (a)
detecting and quantifying the level of expression in a tissue sample of c-
myc, E2F-1 and p21 genes; wherein differential expression of the c-myc,
E2F-1 and p21 genes is indicative of non small cell lung cancer.
In another aspect, the present invention relates to a method of
detecting the progression of non small cell lung cancer in a patient,
comprising: (a) detecting and quantifying the level of expression in a tissue
sample of c-myc, E2F-1 and p21 gene's; wherein differential expression of
the c-myc, E2F-1 and p21 genes is indicative of non small cell lung cancer
progression.
In still other aspects, the present invention relates to a method of
monitoring the treatment of a patient with non small cell lung cancer,
comprising: (a) administering a pharmaceutical composition to the patient;
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(b) preparing a gene expression profile from a cell or tissue sample from the
patient; and (c) comparing the patient gene expression profile to a gene
expression from a cell population selected from the group consisting of
normal lung cells, and non small cell lung cancer.
5 In still more aspects, the present invention relates to a method of
treating a patient with non small cell lung cancer, comprising: (a)
administering to the patient a pharmaceutical composition, wherein the
composition alters the expression of at least one gene in Tables 1 and 5 or
c-myc, E2F-1 and p21 genes; (b) preparing an IGEI comprising
10 standardized gene expression values using StaRT-PCR from a cell or
tissue
sample comprising tumor cells obtained before treatment and another
sample obtained after treatment; and (c) comparing the sample obtained
prior to treatment with the sample obtained after treatment.
Yet other aspects of the present invention relate to a method of
screening for an agent capable of modulating the onset or progression of
non small cell lung cancer, comprising: (a) preparing a first IGEI comprising
standardized gene expression values using StaRT-PCR of a cell population
comprising non small cell cancer cells, wherein the first IGEI determines the
expression level of one or more genes from Tables 1 and 5 or c-myc, E2F-2
and p21 genes; (b) exposing the cell population to the agent; (c) preparing
second IGEI comprising standardized gene expression values using StaRT-
PCR of the agent-exposed cell population; and (d) comparing the first and
second IGEls.
In another aspect, the present invention relates to one or more solid
phase hybridization templates for measuring, in a standardized fashion,
PCR products following standardized quantitative RT-PCR where the
template is formed as follows:
a) preparing at least one solid phase hybridization template where,
for each gene, an oligonucleotide of any length that will bind with
specificity
to both the competitive template, CT, and native template, NT, is spotted to
a filter;
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b) identifying a suitable oligonucleotide such that the region between
the forward primer (common to both the NT and CT) and the 3' 20 bp of the
reverse CT primer is evaluated;
C) attaching an oligonucleotide to a solid support at a previously
designated location;
d) amplifying the CT and NT PCR products and hybridizing to the
spots of the filter wherein each gene (NT and CT) are amplified separately;
e) pooling the PCR products for hybridization; and
f) preparing two oligonucleotide probes, each labeled with a different
fluor, for each gene wherein one oligonucleotide is homologous to, and will
bind to sequences unique to the NT for a gene that was PCR-amplified such
that this oligonucleotide binds to the region of the NT that is not homologous
to the CT and is labeled with a different fluor, and wherein the other
oligonucleotide is specific to the CT and is labeled with a different fluor
such
that this other oligonucleotide is homologous to and will bind to CT
sequences that span the 3' end of the reverse primer. In certain
embodiments, the NT-specific and CT-specific oligonucleotides for multiple
genes are mixed in equal amounts and hybridized to the gene-specific PCR
products bound to the gene-specific oligonucleotides spotted on the filter.
Also, the ratio between the fluors bound to the spot quantify the NT relative
to CT. Although there may be different binding affinities between the CT and
CT probe relative to that between the NT and NT probe, this difference is
consistent between different samples assessed, and from one experiment to
another. It should be noted that the template can comprises at least one
standardized microarray, microbeads, glass slides, or chips prepared by
photolithography, and that the solid support can be a membrane, a glass
support, a filter, a tissue culture dish, a polymeric material, a bead and a
silica support. In certain embodiments, the solid support comprising at least
two oligonucleotides, wherein each of the oligonucleotides comprises a
sequence that specifically hybridizes to at least one gene in Tables 1 and 5
or the c-myc, E2F-1 and p21 genes. It should also be noted that the
oligonucleotides can be covalently attached to the solid support, or
alternatively can be non-covalently attached to the solid support.
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expression level in units of molecules/106 (3-actin molecules for the set of
genes in normal lung tissue.
The invention further includes computer systems comprising a
numerical standardized database containing information identifying the
expression level in lung tissue of a set of genes comprising at least two
genes in Tables 1 and 5 or c-myc x E2F-1/p21; and a user interface to view
the information. In some preferred embodiments, one or more genes may be
selected from a group consisting of the genes listed in Table 5. The
numerical standardized database may further include sequence information
for the genes, information identifying the expression level for the set of
genes in normal lung tissue and malignant tissue (metastatic and
nonmetastatic) and may contain links to external databases such as
GenBank.
The invention further comprises kits useful for the practice of one or
more of the methods of the invention. In some preferred embodiments, a kit
may contain one or more solid supports having attached thereto one or
more oligonucleotides. The solid support may be a high-density
oligonucleotide array. Kits may further comprise one or more reagents for
use with the arrays, one or more signal detection and/or array-processing
instruments, one or more gene expression databases and one or more
analysis and database management software packages. The kits, in certain
preferred embodiments, have StaRT-PCR reagents with reagents to apply
to standardized microarrays.
The invention still further includes methods of using the databases,
such as methods of using the disclosed computer systems to present
information identifying the expression level in a tissue or cell of at least
one
gene in Tables 1 and 5, comprising the step of comparing the expression
level of at least one gene in Tables 1 and 5 in the tissue or cell to the
level
of expression of the gene in the database. In some preferred embodiments,
one or more genes may be selected from a group consisting of the genes
listed in Table 5.
Other features and advantages of the invention will be apparent from
the detailed description and from the claims. Although materials and
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methods similar or equivalent to those described herein can be used in the
practice or testing of the invention, the preferred materials and methods are
described below.
BRIEF DESCRIPTION OF THE DRAWINGS
Figs. la and 1 b are a Table 1 showing primers used for PCR
amplification. Table 1 shows primers used for PCR amplification including
the gene designation, GenBank accession number, Sequence ID number,
primer, sequence, bp position in cDNA, and product length (bp).
The sequences of the expression marker genes are in the public
databases. Tables 1 and 5 provide the GenBank accession number for the
genes. The sequences of the genes in GenBank are equivalent and related
sequences present in GenBank or other public databases. The column
labeled "SEQ ID" refers to the sequence identification number correlating
the listed gene to its sequence information as provided within the sequence
listing of this application.
Fig. 2 is a Table 2 showing the IC 50 for NSCLC cell lines and the
cisplatin levels.
Figs. 3a and 3b are a Table 3 showing the gene expression in
NSCLC cell lines (mRNAs/106ACTB mRNAs).
Fig. 4 is a Table 4 showing the correlation of gene expression with
cisplatin chemoresistance in NSCLC cell lines.
Fig. 5 is a Table 5 showing the statistical assessments of cisplatin
chemoresistance models in NSCLC cell lines.
Fig. 6 is a Table 6 the effect of collection methods on RNA quality in
H1155 human NSCLC cells in Example II which relates to IEGI used for
Fine Needle Analysis (FNA) for lung cancer diagnosis.
Fig. 7 is a Table 7 showing cytological information and diagnosis of
FNA specimen cells in Example.
Fig. 8 is a Table 8 showing gene expression value and index values
for c-myc, E2F-1 and p21 in FNA samples.
Figs. 9a and 9b are schematic illustrations of an analysis of
standardized RT-PCR products with nnicroarrays and microbeads: Fig. 9a
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shows nnicroarrays where the identity of the gene is known by the location of
the microarray; and Fig. 9b shows microbeads where the identity of the
gene is known by the fluorescent color of the bead.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
The present invention is based, in part, on the identification and
quantification of markers that can be used to determine whether cancer cells
are sensitive to a therapeutic agent. Based on these identifications and
quantifications, the present invention provides, without limitation: 1 )
methods for determining whether a therapeutic agent (or combination of
agents) will or will not be effective in stopping or slowing tumor growth; 2)
methods for monitoring the effectiveness of a therapeutic agent (or
combination of agents) used for the treatment of cancer; 3) methods for
identifying new therapeutic agents for the treatment of cancer; 4) methods
for identifying combinations of therapeutic agents for use in treating cancer;
5) methods for identifying specific therapeutic agents and combinations of
therapeutic agents that are effective for the treatment of cancer in specific
patients; and methods for diagnosing cancer.
Definitions
Unless otherwise defined, all technical and scientific terms used
herein have the same meaning as commonly understood by one of ordinary
skill in the art to which this invention belongs. Although methods and
materials similar or equivalent to those described herein can be used in the
practice or testing of the present invention, the preferred methods and
materials are described herein. In the case of conflict, the present
specification, including definitions, will control. In addition, the
materials,
methods, and examples are illustrative only and are not intended to be
limiting.
The articles "a" and "an" are used herein to refer to one or to more
than one (i.e. to at least one) of the grammatical object of the article. By
way
of example, "an element" means one element or more than one element.
A "marker" is a naturally occurring polymer corresponding to at least
one of the nucleic acids listed in Tables 1-5. For example, markers include,
CA 02820020 2013-06-26
without limitation, sense and anti-sense strands of genomic DNA (i.e.
including any introns occurring therein), RNA generated by transcription of
genomic DNA (i.e. prior to splicing), RNA generated by splicing of RNA
transcribed from genomic DNA, and proteins generated by translation of
5 spliced RNA
(i.e. including proteins both before and after cleavage of
normally cleaved regions such as transmembrane signal sequences). As
used herein, "marker" may also include a cDNA made by reverse
transcription of an RNA generated by transcription of genomic DNA
(including spliced RNA).
10 The term
"probe" refers to any molecule which is capable of
selectively binding to a specifically intended target molecule, for example a
marker of the invention. Probes can be, either synthesized by one skilled in
the art, or derived from appropriate biological preparations. For purposes of
detection of the target molecule, probes may be specifically designed to be
15 labeled, as
described herein. Examples of molecules that can be utilized as
probes include, but are not limited to, RNA, DNA, proteins, antibodies, and
organic monomers.
The "normal" level of expression of a marker is the level of
expression of the marker in cells of a patient not afflicted with cancer.
As used herein, the term "promoter/regulatory sequence" means a
nucleic acid sequence which is required for expression of a gene product
operably linked to the promoter/regulatory sequence. In some instances, this
sequence may be the core promoter sequence and in other instances, this
sequence may also include an enhancer sequence and other regulatory
elements which are required for expression of the gene product. The
promoter/regulatory sequence may, for example, be one which expresses
the gene product in a tissue-specific manner.
A "constitutive" promoter is a nucleotide sequence which, when
operably linked with a polynucleotide which encodes or specifies a gene
product, causes the gene product to be produced in a living human cell
under most or all physiological conditions of the cell.
A "transcribed polynucleotide" is a polynucleotide (e.g. an RNA, a
cDNA, or an analog of one of an RNA or cDNA) which is complementary to
CA 02820020 2013-06-26
16
or homologous with all or a portion of a mature RNA made by transcription
of a genomic DNA corresponding to a marker of the invention and normal
post-transcriptional processing (e.g. splicing), if any, of the transcript.
"Complementary" refers to the broad concept of sequence
complementarity between regions of two nucleic acid strands or between
two regions of the same nucleic acid strand. It is known that an adenine
residue of a first nucleic acid region is capable of forming specific hydrogen
bonds ("base pairing") with a residue of a second nucleic acid region which
is antiparallel to the first region if the residue is thymine or uracil.
Similarly, it
is known that a cytosine residue of a first nucleic acid strand is capable of
base pairing with a residue of a second nucleic acid strand which is
antiparallel to the first strand if the residue is guanine. A first region of
a
nucleic acid is complementary to a second region of the same or a different
nucleic acid if, when the two regions are arranged in an antiparallel fashion,
at least one nucleotide residue of the first region is capable of base pairing
with a residue of the second region. Preferably, the first region comprises a
first portion and the second region comprises a second portion, whereby,
when the first and second portions are arranged in an antiparallel fashion, at
least about 50%, and preferably at least about 75%, at least about 90%, or
at least about 95% of the nucleotide residues of the first portion are capable
of base pairing with nucleotide residues in the second portion. More
preferably, all nucleotide residues of the first portion are capable of base
pairing with nucleotide residues in the second portion.
"Homologous" as used herein, refers to nucleotide sequence
similarity between two regions of the same nucleic acid strand or between
regions of two different nucleic acid strands. When a nucleotide residue
position in both regions is occupied by the same nucleotide residue, then
the regions are homologous at that position. A first region is homologous to
a second region if at least one nucleotide residue position of each region is
occupied by the same residue. Homology between two regions is expressed
in terms of the proportion of nucleotide residue positions of the two regions
that are occupied by the same nucleotide residue. Preferably, the first region
comprises a first portion and the second region comprises a second portion,
CA 02820020 2013-06-26
17
whereby, at least about 50%, and preferably at least about 75%, at least
about 90%, or at least about 95% of the nucleotide residue positions of each
of the portions are occupied by the same nucleotide residue. More
preferably, all nucleotide residue positions of each of the portions are
occupied by the same nucleotide residue.
A marker is "fixed" to a substrate if it is covalently or non-covalently
associated with the substrate such the 'substrate can be rinsed with a fluid
(e.g. standard saline citrate, pH 7.4) without a substantial fraction of the
marker dissociating from the substrate.
As used herein, a "naturally-occurring" nucleic acid molecule refers to
an RNA or DNA molecule having a nucleotide sequence that occurs in
nature (e.g. encodes a natural protein).
Cancer is "inhibited" if at least one symptom of the cancer is
alleviated, terminated, slowed, or prevented. As used herein, cancer is also
"inhibited" if recurrence or metastasis of the cancer is reduced, slowed,
delayed, or prevented. Cancer is also inhibited or the cell proliferation
decreases or the cell death rate increases
A "kit" is any manufacture (e.g. a package or container) comprising at
least one reagent, e.g. a probe, for specifically detecting a marker of the
invention, the manufacture being promoted, distributed, or sold as a unit for
performing the methods of the present invention.
Specific Embodiments
The examples provided below concern the identification and
quantification of markers that distinguish in cancer cell lines that are
sensitive to defined chemotherapeutic agents, namely platinum compounds
from those that are not responsive. Accordingly, one or more of the markers
can be used to identify cancer cells that can be successfully treated by that
agent. A change in the expression in one or more of the markers can also
be used to identify cancer cells that cannot be successfully treated by that
agent. These markers can therefore be used in methods for identifying
cancers that have become or are at risk of becoming refractory to treatment
with the agent.
CA 02820020 2013-06-26
18
The expression level of the identified markers may be used to: 1 )
determine if a cancer can be treated by an agent or combination of agents;
2) determine if a cancer is responding to treatment with an agent or
combination of agents; 3) select an appropriate agent or combination of
agents for treating a cancer; 4) monitor the effectiveness of an ongoing
treatment; and 5) identify new cancer treatments (either single agent or
combination of agents).
In particular, the identified markers may be utilized to determine
appropriate therapy, to monitor clinical therapy and human trials of a drug
to being tested
for efficacy, and to develop new agents and therapeutic
combinations.
Accordingly, the present invention provides methods for determining
whether an agent can be used to inhibit cancer cells, comprising the steps
of:
a) obtaining a sample of cancer cells;
b) determining and quantifying the level of expression in the cancer
cells of a marker identified in Tables 1 and 5; and
C) identifying that an agent can. be used to inhibit the cancer cells
when the marker is expressed at a certain level.
The present invention also provides methods for determining whether
an agent is effective in treating cancer, comprising the steps of:
a) obtaining a sample of cancer cells;
b) exposing the sample to an agent;
c) determining and quantifying the level of expression of a marker
identified in Tables 1 and 5 in the sample exposed to the agent and in a
sample that is not exposed to the agent; and
d) identifying that an agent is effective in treating cancer when
expression of the marker is altered in the presence of the agent.
The present invention further provides methods for determining
whether treatment with an agent should be continued in a cancer patient,
comprising the steps of:
a) obtaining two or more samples comprising cancer cells from a
patient during the course of treatment with the agent;
CA 02820020 2013-06-26
19
b) determining and quantifying the level of expression of a marker
identified in Tables 1 and 5 in the two or more samples; and
c) continuing treatment when the expression level of the marker is at
a certain level, e.g., not significantly altered during the course of
treatment.
The present invention also provides methods of identifying new
cancer treatments, comprising the steps of:
a) obtaining a sample of cancer cells;
b) determining and quantifying the level of expression of a marker
identified in Tables 1 and 5;
c) exposing the sample to the cancer treatment;
d) determining the level of expression of the marker in the sample
exposed to the cancer treatment; and
e) identifying that the cancer treatment is effective in treating cancer
when the marker is expressed at a certain level.
Accordingly, in another aspect, the present invention provides
methods for diagnosing cancer, comprising the steps of:
a) obtaining a sample of tissue that might contain cancer cells; and
b) determining and quantifying the level of expression in the tissue
the c-mcy x E2F-1/p21 index.
As used herein, an agent is said to reduce the rate of growth of
cancer cells when the agent can reduce at least 50%, preferably at least
75%, most preferably at least 95% of the growth of the cancer cells. Such
inhibition can further include a reduction in survivability and an increase in
the rate of death of the cancer cells. The amount of agent used for this
determination will vary based on the agent selected. Typically, the amount
will be a predefined therapeutic amount.
As used herein, the term "agent" is defined broadly as anything that
cancer cells may be exposed to in a therapeutic protocol. In the context of
the present invention, such agents include, but are not limited to,
chemotherapeutic agents, such as anti-metabolic agents, e.g., cross-linking
agents, e.g., cisplatin and CBDCA, radiation and ultraviolet light.
Further to the above, the language "chemotherapeutic agent" is
intended to include chemical reagents which inhibit the growth of
CA 02820020 2013-06-26
proliferating cells or tissues wherein the growth of such cells or tissues is
undesirable.
The agents tested in the present methods can be a single agent or a
combination of agents. For example, the present methods can be used to
5 determine whether a single chemotherapeutic agent, such as cisplatin, can
be used to treat a cancer or whether a combination of two or more agents
can be used. Preferred combinations will include agents that have different
mechanisms of action, e.g., the use of an anti-mitotic agent in combination
with an alkylating agent.
10 As used
herein, cancer cells refer to cells that divide at an abnormal
(increased) rate. In particular, the cancer cells include, but are not limited
to,
non-small cell lung cancer (NSCLC). The source of the cancer cells used in
the present method will be based on how the method of the present
invention is being used. For example, if the method is being used to
15 determine
whether a patient's cancer can be treated with an agent, or a
combination of agents, then the preferred source of cancer cells will be
cancer cells obtained from a cancer biopsy from the patient. Alternatively, a
cancer cell line similar to the type of cancer being treated can be assayed.
For example if non-small cell lung cancer (NSCLC) is being treated, then a
20 (NSCLC)
cell line can be used. If the method is being used to monitor the
effectiveness of a therapeutic protocol, then a tissue sample from the patient
being treated is the preferred source. If the method is being used to identify
new therapeutic agents or combinations, any cancer cells, e.g., cells of a
cancer cell line, can be used.
A skilled artisan can readily select and obtain the appropriate cancer
cells that are used in the present method. For cancer cell lines, sources
such as The National Cancer Institute, for the NCI cells used in the
examples, are preferred. For cancer cells obtained from a patient, standard
biopsy methods, such as a needle biopsy, can be employed, taking
necessary precautions known in the art to preserve mRNA integrity.
In the methods of the present invention, the level or amount of
expression of one or more markers selected from the group consisting of the
markers identified in Tablel is determined. As used herein, the level or
CA 02820020 2013-06-26
21
amount of expression refers to the level of expression of an mRNA encoded
by the gene or the level of expression of the protein encoded by the gene
(i.e., whether or not expression is or is not occurring in the cancer cells).
It
also may refer to the values of the interactive gene expression indices (IGEI)
disclosed herein. A skilled artisan can readily adapt known mRNA detection
methods for use in detecting the level of mRNA encoded by one or more of
the (IGEI) marker sets of the present invention.
Proteins from cancer cells can be isolated using techniques that are
well known to those of skill in the art. The protein isolation methods
3.0 employed can, for example, be such as those described in Harlow and
Lane
(Harlow and Lane, 1988, Antibodies: A Laboratory Manual, Cold Spring
Harbor Laboratory Press, Cold Spring Harbor, N.Y.).
A variety of formats can be employed to determine whether a sample
contains a protein that binds to a given antibody. Examples of such formats
include, but are not limited to, enzyme immunoassay (EIA),
radioinnmunoassay (RIA), Western blot analysis and enzyme linked
immunoabsorbant assay (ELISA). A skilled artisan can readily adapt known
protein/antibody detection methods for use in determining whether cancer
cells expresses a protein encoded by one or more of the (IGEI) marker sets
of the present invention.
In one format, antibodies, or antibody fragments, can be used in
methods such as Western blots or immunofluorescence techniques to detect
the expressed proteins. In such uses, it.is generally preferable to immobilize
either the antibody or protein on a solid support. Suitable solid phase
supports or carriers include any support capable of binding an antigen or an
antibody. Well-known supports or carriers include glass, polystyrene,
polypropylene, polyethylene, dextran, nylon, amylases, natural and modified
celluloses, polyacrylamides, gabbros, and magnetite. In addition, the solid
support can be selected from a membrane, a glass support, a filter, a tissue
culture dish, a polymeric material, a bead and a silica support.
In certain embodiments, the solid support comprising at least two
oligonucleotides, wherein each of the oligonucleotides comprises a
sequence that specifically hybridizes to at least one gene in Tables 1 and 5.
CA 02820020 2013-06-26
22
Also, the solid support can include oligonucleotides that are covalently
attached to the solid support, or alternatively, are non-covalently attached
to
the solid support.
One skilled in the art will know many other suitable carriers for
binding antibody or antigen, and will be able to adapt such support for use
with the present invention. For example, protein isolated from cancer cells
can be run on a polyacrylamide gel electrophoresis and immobilized onto a
solid phase support such as nitrocellulose. The support can then be washed
with suitable buffers followed by treatment with the detectably labeled
1.0 marker product specific antibody. The solid phase support can then be
washed with the buffer a second time to remove unbound antibody. The
amount of bound label on the solid support can then be detected by
conventional means.
Another embodiment of the present invention includes a step of
detecting whether an agent stimulates the expression of one or more of the
(IGEI) marker sets of the present invention. Although some of the present
GED marker sets can be expressed in non-treated cancer cells, treatment
with an agent may, or may not, alter expression. Alterations in the
expression level of the GED marker sets of the present invention can
provide a further indication as to whether an agent will or will not be
effective
at reducing the growth rate of the cancer cells.
In such a use, the present invention provides methods for
determining whether an agent, e.g., a chemotherapeutic agent, can be used
to inhibit cancer cells comprising the steps of:
a) obtaining a sample of cancer cells;
b) exposing the sample of cancer cells to one or more test agents;
c) determining and quantifying the level of expression in the cancer
cells of one or more markers selected from the group consisting of the
markers identified in Tablel in the sample exposed to the agent and in a
sample of cancer cells that is not exposed to the agent; and
d) identifying that an agent can be used to treat the cancer when the
expression of one or more of the markers is increased in the presence of
CA 02820020 2013-06-26
23
said agent and/or when the expression of one or more of the markers is not
increased in the presence of said agent.
This embodiment of the methods of the present invention involves the
step of exposing the cancer cells to an agent. The method used for
exposing the cancer cells to the agent will be based primarily on the source
and nature of the cancer cells and the agent being tested. The contacting
can be performed in vitro or in vivo, in a patient being treated/evaluated or
in
animal model of a cancer. For cancer cells and cell lines and chemical
compounds, exposing the cancer cells involves contacting the cancer cells
with the compound, such as in tissue culture media. A skilled artisan can
readily adapt an appropriate procedure for contacting cancer cells with any
particular agent or combination of agents.
As discussed above, the identified (IGEI) marker sets can also be
used to assess whether a tumor has become refractory to an ongoing
treatment (e.g., a chemotherapeutic treatment). When a tumor is no longer
responding to a treatment the expression profile of the tumor cells will
change: the level of expression of one or more of the markers will be
reduced and/or the level of expression of one or more of the markers will
increase.
In such a use, the invention provides methods for determining
whether an anti-cancer treatment should be continued in a cancer patient,
comprising the steps of:
a) obtaining two or more samples of cancer cells from a patient
undergoing anti-cancer therapy;
b) determining and quantifying the level of expression of one or more
markers selected from the group and one or more of the corresponding
(IGEI) marker sets in the sample exposed to the agent and in a sample of
cancer cells that is not exposed to the agent; and
c) discontinuing treatment when the expression of one or more (IGEI)
marker sets is altered.
As used herein, a patient refers to any subject undergoing treatment
for cancer. The preferred subject will be a human patient undergoing
chemotherapy treatment.
CA 02820020 2013-06-26
24
This embodiment of the present invention relies on comparing two or
more samples obtained from a patient undergoing anti-cancer treatment. In
general, it is preferable to obtain a first sample from the patient prior to
beginning therapy and one or more samples during treatment. In such a
use, a baseline of expression prior to therapy is determined and then
changes in the baseline state of expression are monitored during the course
of therapy. Alternatively, two or more successive samples obtained during
treatment can be used without the need of a pre-treatment baseline sample.
In such a use, the first sample obtained from the subject is used as a
baseline for determining whether the expression of a particular marker is
increasing or decreasing.
In general, when monitoring the effectiveness of a therapeutic
treatment, two or more samples from the patient are examined. Preferably,
three or more successively obtained samples are used, including at least
one pretreatment sample.
The present invention further provides kits comprising
compartmentalized containers comprising reagents for detecting one or
more, preferably two or more, of the markers and/or(IGEI) marker sets of the
present invention. As used herein a kit is defined as a pre-packaged set of
containers into which reagents are placed. The reagents included in the kit
comprise probes/primers and/or antibodies for use in detecting GED
marker sets expression. In addition, the kits of the present invention may
preferably contain instructions which describe a suitable detection assay.
Such kits can be conveniently used, e.g., in clinical settings, to diagnose
patients exhibiting symptoms of cancer.
Various aspects of the invention are described in further detail in the
following subsections.
Nucleic Acid Samples
It is apparent to one of ordinary skill in the art, nucleic acid samples
used in the methods and assays of the invention may be prepared by any
available method or process. Methods of isolating total mRNA are also well
known to those of skill in the art. Such samples include RNA samples, but
also include cDNA synthesized from an mRNA sample isolated from a cell
CA 02820020 2013-06-26
or tissue of interest. Such samples also include DNA amplified from the
cDNA, and an RNA transcribed from the amplified DNA. One of skill in the
art would appreciate that it is desirable to inhibit or destroy RNase present
in
homogenates before homogenates can be used.
5 Biological
samples may be of any biological tissue or fluid or cells
from any organism as well as cells raised in vitro, such as cell lines and
tissue culture cells. Frequently the sample will be a "clinical sample" which
is
a sample derived from a patient. Typical clinical samples include, but are not
limited to, sputum, blood, blood-cells (e.g., white cells), tissue or fine
needle
10 biopsy
samples, urine, peritoneal fluid, and pleural fluid, or cells therefrom.
Biological samples may also include sections of tissues, such as frozen
sections or formalin fixed sections taken for histological purposes.
Thus, one aspect of the invention pertains to isolated nucleic acid
molecules that correspond to a marker of the invention, including nucleic
15 acids which encode a polypeptide corresponding to a marker of the
invention or a portion of such a polypeptide. Isolated nucleic acids of the
invention also include nucleic acid molecules sufficient for use as
hybridization probes to identify nucleic acid molecules that correspond to a
marker of the invention, including nucleic acids which encode a polypeptide
20 corresponding
to a marker of the invention, and fragments of such nucleic
acid molecules, e.g., those suitable for use as PCR primers for the
amplification or mutation of nucleic acid molecules. As used herein, the term
"nucleic acid molecule" is intended to include DNA molecules (e.g., cDNA or
genomic DNA) and RNA molecules (e.g., mRNA) and analogs of the DNA or
25 RNA generated
using nucleotide analogs. The nucleic acid molecule can be
single-stranded or double-stranded, but ,preferably is double-stranded DNA.
An "isolated" nucleic acid molecule is one which is separated from
other nucleic acid molecules which are present in the natural source of the
nucleic acid molecule. Preferably, an "isolated" nucleic acid molecule is free
of sequences (preferably protein-encoding sequences) which naturally flank
the nucleic acid (i.e., sequences located at the 5' and 3' ends of the nucleic
acid) in the genomic DNA of the organism from which the nucleic acid is
derived.
CA 02820020 2013-06-26
=
26
A nucleic acid molecule of the present invention, e.g., a nucleic acid
encoding a protein corresponding to a marker listed in Table 1, can be
isolated using standard molecular biology techniques and the sequence
information in the database records described herein. Using all or a portion
of such nucleic acid sequences, nucleic acid molecules of the invention can
be isolated using standard hybridization and cloning techniques (e.g., as
described in Sambrook et al., ed., Molecular Cloning: A Laboratory Manual,
2nd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.,
1989).
3.0 A nucleic acid
molecule of the invention can be amplified using
cDNA, mRNA, or genomic DNA as a template and appropriate
oligonucleotide primers according to standard PCR amplification techniques.
The nucleic acid so amplified can be cloned into an appropriate vector and
characterized by DNA sequence analysis. Furthermore, oligonucleotides
corresponding to all or a portion of a nucleic acid molecule of the invention
can be prepared by standard synthetic techniques, e.g., using an automated
DNA synthesizer.
In another preferred embodiment, an isolated nucleic acid molecule
of the invention comprises a nucleic acid molecule which has a nucleotide
sequence complementary to the nucleotide sequence of a nucleic acid
corresponding to a marker of the invention or to the nucleotide sequence of
a nucleic acid encoding a protein which corresponds to a marker of the
invention. A nucleic acid molecule which is complementary to a given
nucleotide sequence is one which is sufficiently complementary to the given
nucleotide sequence that it can hybridize to the given nucleotide sequence
thereby forming a stable duplex.
Moreover, a nucleic acid molecule of the invention can comprise only
a portion of a nucleic acid sequence, wherein the full length nucleic acid
sequence comprises a marker of the invention or which encodes a
polypeptide corresponding to a marker of the invention. Such nucleic acids
can be used, for example, as a probe or primer. The probe/primer typically is
used as one or more substantially purified oligonucleotides. The
oligonucleotide typically comprises a region of nucleotide sequence that
CA 02820020 2013-06-26
27
hybridizes under stringent conditions to at least about 7, preferably about 12
or more consecutive nucleotides of a nucleic acid of the invention.
Probes based on the sequence of a nucleic acid molecule of the
invention can be used to detect transcripts or genomic sequences
corresponding to one or more markers of the invention. The probe
comprises a label group attached thereto, e.g., a radioisotope, a fluorescent
compound, an enzyme, or an enzyme co-factor. Such probes can be used
as part of a diagnostic test kit for identifying cells or tissues which mis-
express the protein, such as by measuring levels of a nucleic acid molecule
encoding the protein in a sample of cells from a subject, e.g., detecting
mRNA levels or determining whether a gene encoding the protein has been
mutated or deleted.
The invention further encompasses nucleic acid molecules that differ,
due to degeneracy of the genetic code, from the nucleotide sequence of
nucleic acids encoding a protein which corresponds to a marker of the
invention, and thus encode the same protein.
In addition to the nucleotide sequences described in the GenBank
database records described herein, it will be appreciated by those skilled in
the art that DNA sequence polymorphisms that lead to changes in the amino
acid sequence can exist within a population (e.g., the human population).
Such genetic polymorphisms can exist among individuals within a population
due to natural allelic variation. An allele is one of a group of genes which
occur alternatively at a given genetic locus. In addition, it will be
appreciated
that DNA polymorphisms that affect RNA expression levels can also exist
that may affect the overall expression level of that gene (e.g., by affecting
regulation or degradation).
As used herein, the phrase "allelic variant" refers to a nucleotide
sequence which occurs at a given locus or to a polypeptide encoded by the
nucleotide sequence.
As used herein, the terms "gene" and "recombinant gene" refer to
nucleic acid molecules comprising an open reading frame encoding a
polypeptide corresponding to a marker of the invention. Such natural allelic
variations can typically result in 1-5% variance in the nucleotide sequence of
CA 02820020 2013-06-26
28
a given gene. Alternative alleles can be identified by sequencing the gene of
interest in a number of different individuals. This can be readily carried out
by using hybridization probes to identify the same genetic locus in a variety
of individuals. Any and all such nucleotide variations and resulting amino
acid polymorphisms or variations that are the result of natural allelic
variation and that do not alter the functional activity are intended to be
within
the scope of the invention.
In another embodiment, an isolated nucleic acid molecule of the
invention is at least 7, 15, 20, 25, 30, 40, 60, 80, 100, 150, 200, 250, 300,
350, 400, 450, 550, 650, 700, 800, 900, 1000, 1200, 1400, 1600, 1800,
2000, 2200, 2400, 2600, 2800, 3000, 3500, 4000, 4500, or more nucleotides
in length and hybridizes under stringent conditions to a nucleic acid
corresponding to a marker of the invention or to a nucleic acid encoding a
protein corresponding to a marker of the invention. As used herein, the term
"hybridizes under stringent conditions" is intended to describe conditions for
hybridization and washing under which nucleotide sequences at least 60%
(65%, 70%, preferably 75%) identical to each other typically remain
hybridized to each other. Such stringent conditions are known to those
skilled in the art and can be found in sections 6.3.1-6.3.6 of Current
Protocols in Molecular Biology, John Wiley & Sons, N.Y. (1989). A preferred,
non-limiting example of stringent hybridization conditions are hybridization
in
6× sodium chloride/sodium citrate (SSC) at about 45 degree C,
followed by one or more washes in 0.2× SSC, 0.1% SOS at 50-65
degree C
In addition to naturally-occurring allelic variants of a nucleic acid
molecule of the invention that can exist in the population, the skilled
artisan
will further appreciate that sequence changes can be introduced by mutation
thereby leading to changes in the amino acid sequence of the encoded
protein, without altering the biological activity of the protein encoded
thereby. For example, one can make nucleotide substitutions leading to
amino acid substitutions at "non-essential" amino acid residues. A "non-
essential" amino acid residue is a residue that can be altered from the wild-
type sequence without altering the biological activity, whereas an "essential"
CA 02820020 2013-06-26
29
amino acid residue is required for biological activity. For example, amino
acid residues that are not conserved or only semi-conserved among
homologs of various species may be non-essential for activity and thus
would be likely targets for alteration. Alternatively, amino acid residues
that
are conserved among the homologs of various species (e.g., murine and
human) may be essential for activity and thus would not be likely targets for
alteration.
Accordingly, another aspect of the invention pertains to nucleic acid
molecules encoding a polypeptide of the invention that contain changes in
amino acid residues that are not essential for activity. Such polypeptides
differ in amino acid sequence from the naturally-occurring proteins which
correspond to the markers of the invention, yet retain biological activity. In
one embodiment, such a protein has an amino acid sequence that is at least
about 40% identical, 50%, 60%, 70%, 80%, 90%, 95%, or 98% identical to
the amino acid sequence of one of the proteins which correspond to the
markers of the invention.
An isolated nucleic acid molecule encoding a variant protein can be
created by introducing one or more nucleotide substitutions, additions or
deletions into the nucleotide sequence of nucleic acids of the invention,
such that one or more amino acid residue substitutions, additions, or
deletions are introduced into the encoded protein. Mutations can be
introduced by standard techniques, such as site-directed mutagenesis and
PCR-mediated mutagenesis. Preferably, conservative amino acid
substitutions are made at one or more predicted non-essential amino acid
residues. A "conservative amino acid substitution" is one in which the amino
acid residue is replaced with an amino acid residue having a similar side
chain. Families of amino acid residues having similar side chains have been
defined in the art. These families include amino acids with basic side chains
(e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid,
glutamic acid), uncharged polar side = chains (e.g., glycine, asparagine,
glutamine, serine, threonine, tyrosine, cysteine), non-polar side chains
(e.g.,
alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine,
tryptophan), beta-branched side chains (e.g., threonine, valine, isoleucine)
CA 02820020 2013-06-26
30.
and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan,
histidine). Alternatively, mutations can be introduced randomly along all or
part of the coding sequence, such as by saturation mutagenesis, and the
resultant mutants can be screened for biological activity to identify mutants
that retain activity. Following mutagenesis, the encoded protein can be
expressed recombinantly and the activity of the protein can be determined.
The present invention encompasses antisense nucleic acid
molecules, i.e., molecules which are complementary to a sense nucleic acid
of the invention, e.g., complementary to the coding strand of a double-
stranded cDNA molecule corresponding to a marker of the invention or
complementary to an mRNA sequence corresponding to a marker of the
invention. Accordingly, an antisense nucleic acid of the invention can
hydrogen bond to (i.e. anneal with) a sense nucleic acid of the invention.
The antisense nucleic acid can be complementary to an entire coding
strand, or to only a portion thereof, e.g., all or part of the protein coding
region (or open reading frame). An antisense nucleic acid molecule can also
be antisense to all or part of a non-coding region of the coding strand of a
nucleotide sequence encoding a polypeptide of the invention. The non-
coding regions ("5' and 3' untranslated regions") are the 5' and 3' sequences
which flank the coding region and are not translated into amino acids.
An antisense oligonucleotide can be, for example, about 5, 10, 15,
20, 25, 30, 35, 40, 45, or 50 or more nucleotides in length. An antisense
nucleic acid of the invention can be constructed using chemical synthesis
and enzymatic ligation reactions using procedures known in the art. For
example, an antisense nucleic acid (e.g., an antisense oligonucleotide) can
be chemically synthesized using naturally occurring nucleotides or variously
modified nucleotides designed to increase the biological stability of the
molecules or to increase the physical stability of the duplex formed between
the antisense and sense nucleic acids, e.g., phosphorothioate derivatives
and acridine substituted nucleotides can be used. Examples of modified
nucleotides which can be used to generate the antisense nucleic acid
include 5-fluorouracil, 5-bromouracil, 5-
chlorouracil, 5-iodouracil,
hypoxanthine, xanthine, 4-acetylcytosine, 5-(carboxyhydroxylmethyl) uracil,
CA 02820020 2013-06-26
31
5-carboxymethylaminomethy1-2-thiouridin-e, 5 carboxymethylaminomethyl-
uracil, dihydrouracil, beta-D-galactosylqueosine, inosine, N6-isopentenyl-
adenine, 1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-
methyladenine, 2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-
adenine, 7-methylguanine, 5-methylaminomethyluracil, 5-methoxyamino-
methy1-2-thiour- acil, beta-D-nnannosylqueosine, 5'-methoxycarboxy-
methyluracil, 5-methoxyuracil, 2-nnethylthio-N6-isopentenyladenine, uracil-5-
oxyacetic acid (v), wybutoxosine, pseudouracil, queosine, 2-thiocytosine, 5-
methy1-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-nnethyluracil, uracil-5-
acid methylester, uracil-5-oxyacetic acid (v), 5-methyl-2-thiouracil,
3-(3-amino-3-N-2-carboxypropyl) uracil, (acp3)w, and 2,6-diaminopurine.
Alternatively, the antisense nucleic acid can be produced biologically using
an expression vector into which a nucleic acid has been sub-cloned in an
antisense orientation (i.e., RNA transcribed from the inserted nucleic acid
will be of an antisense orientation to a target nucleic acid of interest,
described further in the following subsection).
The antisense nucleic acid molecules of the invention are typically
administered to a subject or generated in situ such that they hybridize with
or bind to cellular mRNA and/or genomic DNA encoding a polypeptide
corresponding to a selected marker of the invention to thereby inhibit
expression of the marker, e.g., by inhibiting transcription and/or
translation.
The hybridization can be by conventional nucleotide complementarity to
form a stable duplex, or, for example, in the case of an antisense nucleic
acid molecule which binds to DNA duplexes, through specific interactions in
the major groove of the double helix. Examples of a route of administration
of antisense nucleic acid molecules of the invention includes direct injection
at a tissue site or infusion of the antisense nucleic acid into an ovary-
associated body fluid. Alternatively, antisense nucleic acid molecules can be
modified to target selected cells and then administered systemically. For
example, for systemic administration, antisense molecules can be modified
such that they specifically bind to receptors or antigens expressed on a
selected cell surface, e.g., by linking the antisense nucleic acid molecules
to
peptides or antibodies which bind to cell surface receptors or antigens. The
CA 02820020 2013-06-26
32
antisense nucleic acid molecules can also be delivered to cells using the
vectors described herein. To achieve sufficient intracellular concentrations
of
the antisense molecules, vector constructs in which the antisense nucleic
acid molecule is placed under the control of a strong pol II or pol Ill
promoter
are preferred.
The invention also encompasses ribozymes. Ribozymes are catalytic
RNA molecules with ribonuclease activity which are capable of cleaving a
single-stranded nucleic acid, such as. an mRNA, to which they have a
complementary region. Thus, ribozymes can be used to catalytically cleave
mRNA transcripts to thereby inhibit translation of the protein encoded by the
mRNA. A ribozynne having specificity for a nucleic acid molecule encoding a
polypeptide corresponding to a marker of the invention can be designed
based upon the nucleotide sequence of a cDNA corresponding to the
marker.
The invention also encompasses nucleic acid molecules which form
triple helical structures. For example, expression of a polypeptide of the
invention can be inhibited by targeting nucleotide sequences
complementary to the regulatory region of the gene encoding the
polypeptide (e.g., the promoter and/or enhancer) to form triple helical
structures that prevent transcription of the gene in target cells.
The invention also encompasses the use of RNA interference or
"RNAi" which is a term initially coined by Fire and co-workers to describe the
observation that double-stranded RNA (dsRNA) can block gene expression
when it is introduced into worms (Fire et al. (1998) Nature 391, 806-811).
dsRNA directs gene-specific, post-transcriptional silencing in many
organisms, including vertebrates, and has provided a new tool for studying
gene function.
The phenomenon of RNA interference is described and discussed in
Bass, Nature 411 : 428-29 (2001 ); Elbashir et al., Nature 411 : 494-98
(2001); and Fire et al., Nature 391 : 806-11 (1998), where methods of
making interfering RNA also are discussed. An "siRNA" or "RNAi" refers to a
nucleic acid that forms a double stranded RNA, which double stranded RNA
has the ability to reduce or inhibit expression of a gene or target gene when
CA 02820020 2013-06-26
33
the siRNA expressed in the same cell as the gene or target gene. "siRNA"
thus refers to the double stranded RNA formed by the complementary
strands. The complementary portions of the siRNA that hybridize to form the
double stranded molecule typically have substantial or complete identity. In
complete identity to a target gene and forms a double stranded siRNA. The
sequence of the siRNA can correspond to the full-length target gene, or a
subsequence thereof. Typically, the siRNA is at least about 15-50
nucleotides in length (e.g., each complementary sequence of the double
siRNA is about 15-50 base pairs in length, preferable about preferably about
20-30 base nucleotides, preferably about 20-25 nucleotides in length, e.g.,
20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides in length.
In various embodiments, the nucleic acid molecules of the invention
improve, e.g., the stability, hybridization, or solubility of the molecule.
For
example, the deoxyribose phosphate backbone of the nucleic acids can be
modified to generate peptide nucleic acids. As used herein, the terms
"peptide nucleic acids" or "PNAs" refer to nucleic acid mimics, e.g., DNA
20 mimics, in which the deoxyribose phosphate backbone is replaced by a
pseudopeptide backbone and only the four natural nucleobases are
retained. The neutral backbone of PNAs has been shown to allow for
specific hybridization to DNA and RNA under conditions of low ionic
strength. The synthesis of PNA oligomers can be performed using standard
PNAs can be used in therapeutic and diagnostic applications. For
example, PNAs can be used as antisense or antigene agents for sequence-
specific modulation of gene expression by, e.g., inducing transcription or
translation arrest or inhibiting replication. PNAs can also be used, e.g., in
the
clamping; as artificial restriction enzymes when used in combination with
other enzymes.
CA 02820020 2013-06-26
34
In other embodiments, the oligonucleotide can include other
appended groups such as peptides (e.g., for targeting host cell receptors in
vivo), or agents facilitating transport across the cell membrane or the blood-
brain barrier. In addition, oligonucleotides can be modified with
hybridization-triggered cleavage agents or intercalating agents. The
oligonucleotide can be conjugated to another molecule, e.g., a peptide,
hybridization triggered cross-linking agent, transport agent, hybridization-
triggered cleavage agent, etc.
The invention also includes molecular beacon nucleic acids having at
least one region which is complementary to a nucleic acid of the invention,
such that the molecular beacon is useful for quantitating the presence of the
nucleic acid of the invention in a sample. A "molecular beacon" nucleic acid
is a nucleic acid comprising a pair of complementary regions and having a
fluorophore and a fluorescent quencher associated therewith. The
fluorophore and quencher are associated with different portions of the
nucleic acid in such an orientation that when the complementary regions are
annealed with one another, fluorescence of the fluorophore is quenched by
the quencher. When the complementary regions of the nucleic acid are not
annealed with one another, fluorescence of the fluorophore is quenched to a
lesser degree.
MICROARRAYS
In another aspect, the present invention describes the use of high
density oligonucleotide microarrays or solid supports or microbeads to
measure in a standardized fashion PCR products following standardized
quantitative RT-PCR according to the methods described herein, as shown
in Figures 9a and 9b.
In certain embodiments, the preparation of high-density
oligonucleotide arrays can be made with the following properties. For each
gene, an oligonucleotide of any length that will bind with specificity to both
the competitive template, CT, and native template, NT, is spotted of a filter.
To identify a suitable oligonucleotide, the region between the forward primer
(common to both the NT and CT) and the 3' 20 bp of the reverse CT primer
is evaluated. An oligonucleotide with high melting temperature, preferably
CA 02820020 2013-06-26
greater than about 70 degrees centigrade, and be attached to the solid
support at a previously designated location In Figure 9a the oligonucleotides
specific to each gene are designated with different bars (open, slashed, or
striped).
5 Then, the CT
and NT PCR products, amplified according to the
methods described above, are hybridized to the spots. Each gene (NT and
CT) is amplified separately. Then the PCR products are pooled for
hybridization to the membrane described above, and illustrated in Figure 9a.
The CT and NT PCR products appear as thin black curved lines in the
10 Figure 9a.
Two oligonucleotide probes, each labeled with a different fluor, are
prepared for each gene. One oligonucleotide will be homologous to, and will
bind to sequences unique to the NT for a gene that was PCR-amplified
using the methods described herein. This oligonucleotide will bind to the
15 region of the
NT that is not homologous to the CT and will be labeled with a
different fluor. The other oligonucleotide will be specific to the CT and will
be
labeled with a different fluor. It will be homologous to and will bind to CT
sequences that span the 3' end of the reverse primer. The NT-specific and
CT-specific oligonucleotides for multiple genes will be mixed in equal
20 amounts and
hybridized to the gene-specific PCR products bound to the
gene-specific oligonucleotides spotted on the filter. The ratio between the
fluors bound to the spot will quantify the NT relative to CT. The fluorescent
tagged probe (shaded black) is specific to the NT and the fluorescent tagged
probe (unshaded) is specific to the CT.
25 In this assay,
although there may be different binding affinities
between the CT and CT probe relative to that between the NT and NT
probe, this difference will be consistent between different samples
assessed, and from one experiment to another.
This method also works with other solid phase hybridization
30 templates including, for example, microbeads, glass slides, or chips
prepared by photolithography. No matter what template is used, the
products of standardized RT-PCT, using the standardized mixture of
competitive templates, will be the starting point, as shown with microbeads
CA 02820020 2013-06-26
36
in Figure 9b where microbeads gene specificity is conferred by the
fluorescent color of the bead, rather than the location on the microarray.
CISPLATIN
The examples set forth below relate to cis-Diamminedichloroplatinum
(II), otherwise known as cisplatin, and related compounds. Cisplatin is a
chemical compound within a family of platinum coordination complexes
which are art-recognized as being a family of related compounds. Cisplatin
was the first platinum compound shown to have anti-malignant properties.
The language "platinum compounds" is intended to include cisplatin,
compounds which are structurally similar to cisplatin, as well as analogs and
derivatives of cisplatin. The language "platinum compounds" can also
include "mimics". "Mimics" is intended to include compounds which may not
be structurally similar to cisplatin but mimic the therapeutic activity of
cisplatin or structurally related compounds in vivo.
The platinum compounds of this invention are those compounds
which are useful for inhibiting tumor growth in subjects (patients). More than
1000 platinum-containing compounds have been synthesized and tested for
therapeutic properties. One of these, carboplatin, has been approved for
treatment of ovarian cancer. Both cisplatin and carboplatin are amenable to
intravenous delivery. However, compounds of the invention can be
formulated for therapeutic delivery by any number of strategies. The term
platinum compounds also is intended to include pharmaceutically
acceptable salts and related compounds. Platinum compounds have
previously been described in U.S. Pat. Nos. 6,001,817, 5,945,122,
5,942,389, 5,922,689, 5,902,610, 5,866,617, 5,849,790, 5,824,346,
5,616,613, and 5,578,571.
Cisplatin and related compounds are thought to enter cells through
diffusion, whereupon the molecule likely undergoes metabolic processing to
yield the active metabolite of the drug, which then reacts with nucleic acids
and proteins. Cisplatin has biochemical properties similar to that of
bifunctional alkylating agents, producing interstrand, intrastrand, and
monofunctional adduct cross-linking with DNA.
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37
DATABASES
The present invention includes relational numerically standardized
databases containing sequence information, for instance for the genes of
Tables 1 and 5, as well as gene expression information in various lung
tissue samples. Databases may also contain information associated with a
given sequence or tissue sample such as descriptive information about the
gene associated with the sequence information, or descriptive information
concerning the clinical status of the tissue sample, or the patient from which
the sample was derived. The database may be designed to include different
parts, for instance a sequences database and a gene expression database.
Methods for the configuration and construction of such databases are widely
available.
The numerically standardized databases of the invention may be
linked to an outside or external database. In a preferred embodiment, as
described in Tables 1-5, the external database is GenBank and the
associated databases maintained by the National Center for Biotechnology
Information (NCBI).
Any appropriate computer platform may be used to perform the
necessary comparisons between sequence information, gene expression
information and any other information in the database or provided as an
input. For example, a large number of computer workstations are available
from a variety of manufacturers, such has those available from Silicon
Graphics. Client-server environments, database servers and networks are
also widely available and appropriate platforms for the databases of the
invention.
The databases of standardized numerical data of the invention may
be used to produce, among other things, electronic Northerns to allow the
user to determine the cell type or tissue in which a given gene is expressed
and to allow determination of the abundance or expression level of a given
gene in a particular tissue or cell.
The databases of the invention may also be used to present
information identifying the expression level in a tissue or cell of a set of
genes comprising at least one gene in Tables 1-5 comprising the step of
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38
comparing the expression level of at least one gene in Tables 1-5 in the
tissue to the level of expression of the gene in the database. Such methods
may be used to predict the physiological state of a given tissue by
comparing the level of expression of a gene or genes in Tables 1-5 from a
sample to the expression levels found in tissue from normal lung, malignant
lung or NSCLC. Such methods may also be used in the drug or agent
screening assays as described below.
COMPUTER SYSTEM
In another aspect, the present invention relates to a computer system
comprising: (a) a database containing standardized numerical gene
expression information identifying the expression level in lung tissue of a
set
of genes comprising at least two genes in Tables 1 and 5 or c-myc x E2F-
a/p21; and (b) a user interface to view the information. The database can
further include at least one or more of the following: sequence information
for the genes; information identifying the expression level for the set of
genes in normal lung tissue; information identifying the expression level of
the set of genes in non small cell cancer tissue, records including
descriptive
information from an external database, which information correlates said
genes to records in the external database; including, for example, where the
external database is GenBank and information or specific characteristics of
the cells or tissues or patients from which the were derived.
In another aspect, the present invention relates to a method of using
the computer system described above to present information identifying the
expression level in a tissue or cell of at least one gene in Tables 1 and 5,
by
comparing the expression level of at least one gene in Tables 1 and 5 in the
tissue or cell to the level of expression of the gene in the database. In
certain embodiments, the expression level of at least two, five, seven, and/or
ten genes are compared.
In yet other aspects, the method further includes displaying the level
of expression of at least one gene in the tissue or cell sample compared to
the expression level in lung cancer.
KITS
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39
The invention further includes kits combining, in different combinations, at
least one of: high-density oligonucleotide arrays, reagents for use with the
microarrays, reagents for StaRT-PCR amplification of the specified genes
including gene specific primers and standardized mixtures of internal
standards, signal detection and array-processing instruments, gene
expression databases, and analysis and database management software
described above. The kits may be used, for example, to predict or model the
toxic response of a test compound, to monitor the progression of disease
states, to identify genes that show promise as new drug targets and to
screen known and newly designed drugs as discussed herein.
In certain embodiments, the kit includes at least one solid support, as
described herein, packaged with gene expression information for said
genes. In certain embodiments, the gene expression information comprises
gene expression levels in a tissue or cell sample exposed to a toxin. Also, in
certain embodiments, the gene expression information is in an electronic
format, including, for example, the standardized gene expression database
described herein.
The databases packaged with the kits are a compilation of expression
patterns from human or laboratory animal genes and gene fragments
(corresponding to the genes of Tables 1 and 5). Data is collected from a
repository of both normal and diseased tissues and provides reproducible,
quantitative results, i.e., the degree to which a gene is up-regulated or
down-regulated under a given condition.
The kits are useful in the pharmaceutical industry, where the need for
early drug testing is strong due to the high costs associated with drug
development, but where bioinformatics, in particular gene expression
informatics, is still lacking. These kits reduce the costs, time and risks
associated with traditional new drug screening using cell cultures and
laboratory animals. The results of large-scale drug screening of pre-grouped
patient populations, pharmacogenomics testing, can also be applied to
select drugs with greater efficacy and fewer side-effects. The kits may also
be used by smaller biotechnology companies and research institutes who do
not have the facilities for performing such large-scale testing themselves.
CA 02820020 2013-06-26
Databases and software designed for use with microarrays is
discussed in Balaban et al., U.S. Pat. No. Nos. 6,229,911, a computer-
implemented method for managing information, stored as indexed tables,
collected from small or large numbers of microarrays, and U.S. Pat. No.
5 6,185,561, a computer-based method with data mining capability for
collecting gene expression level data, adding additional attributes and
reformatting the data to produce answers to various queries. Chee et al.,
U.S. Pat. No. 5,974,164, disclose a software-based method for identifying
mutations in a nucleic acid sequence based on differences in probe
10 fluorescence intensities between wild type and mutant sequences that
hybridize to reference sequences.
ASSAYS and Identification of Therapeutic and Drug Screening Targets
It should be understood that in certain preferred embodiments, the
microarrays as described herein, and in particular, with reference to the
15 example shown in Figure 9a and 9b, are especially useful. However, it
should also be understood, that in certain other embodiments, other
hybridization assay format may be used, including solution-based and solid
support-based assay formats. Solid supports containing oligonucleotide
probes for differentially expressed genes of the invention can be filters,
20 polyvinyl chloride dishes, silicon or glass based chips, etc. Such
wafers and
hybridization methods are widely available. Any solid surface to which
oligonucleotides can be bound, either directly or indirectly, either
covalently
or non-covalently, can be used. Examples of a solid support include a high
density array or DNA chip. These contain a particular oligonucleotide probe
25 in a predetermined location on the array. Each predetermined location may
contain more than one molecule of the probe, but each molecule within the
predetermined location has an identical sequence. Such predetermined
locations are termed features.
There may be, for example, about 2, 10, 100, 1000 to 10,000;
30 100,000 or 400,000 of such features on a single solid support. The solid
support, or the area within which the probes are attached may be on the
order of a square centimeter.
CA 02820020 2013-06-26
41
Oligonucleotide probe arrays for expression monitoring can be made
and used according to any techniques known in the art. Such probe arrays
may contain at least two or more oligonucleotides that are complementary to
or hybridize to two or more of the genes described herein. Such arrays may
also contain oligonucleotides that are complementary or hybridize to at least
about 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 50, 70, 100 or more the genes
described herein.
Methods of forming high density arrays of oligonucleotides with a
minimal number of synthetic steps are known. The oligonucleotide analogue
array can be synthesized on a solid substrate by a variety of methods,
including, but not limited to, light-directed chemical coupling, and
mechanically directed coupling. In brief, the light-directed combinatorial
synthesis of oligonucleotide arrays on a glass surface proceeds using
automated phosphoramidite chemistry and chip masking techniques. In one
specific implementation, a glass surface is derivatized with a silane reagent
containing a functional group, e.g., a hydroxyl or amine group blocked by a
photolabile protecting group. Photolysis through a photolithogaphic mask is
used selectively to expose functional groups which are then ready to react
with incoming 5' photoprotected nucleoside phosphoramidites. The
phosphoramidites react only with those sites which are illuminated (and thus
exposed by removal of the photolabile blocking group). Thus, the
phosphoramidites only add to those areas selectively exposed from the
preceding step. These steps are repeated until the desired array of
sequences have been synthesized on the solid surface. Combinatorial
synthesis of different oligonucleotide analogues at different locations on the
array is determined by the pattern of illumination during synthesis and the
order of addition of coupling reagents.
In addition to the foregoing, additional methods can be used to
generate an array of oligonucleotides on a single substrate. High density
nucleic acid arrays can also be fabricated by depositing premade or natural
nucleic acids in predetermined positions. Synthesized or natural nucleic
acids are deposited on specific locations of a substrate by light directed
targeting and oligonucleotide directed targeting. Another embodiment uses
CA 02820020 2013-06-26
42
a dispenser that moves from region to region to deposit nucleic acids in
specific spots.
DETERMINATION OF IGEI
A sample of cancerous cells with unknown sensitivity to a given drug
is obtained from a patient. An expression level is measured in the sample for
a gene corresponding to one of the nucleotide sequences claimed herein as
a (IGEI) marker set. The expression level of the marker in the sample is
compared with the expression level of the marker measured previously in
cells with known drug sensitivity. If the expression level of the marker in
the
sample is most similar to the expression levels of the marker in cells with
low sensitivity to the given drug, then low sensitivity to that drug is
predicted
for the sample. If the expression level of the marker in the sample is most
similar to the expression levels of the marker in cells with medium
sensitivity
to the given drug, then medium sensitivity to that drug is predicted for the
sample. If the expression level is most similar to the expression levels of
the
marker in cells with high sensitivity to the given drug, then high sensitivity
to
that drug is predicted for the sample.
Thus, by examining the expression of one or more of the identified
markers in a sample of cancer cells, it is possible to determine which
therapeutic agent(s), or combination of agents, to use as the appropriate
treatment agents.
By examining the expression of one or more of the identified markers
in a sample of cancer cells taken from a patient during the course of
therapeutic treatment, it is also possible to determine whether the
therapeutic agent is continuing to work or whether the cancer has become
resistant (refractory) to the treatment protocol. These determinations can be
made on a patient-by-patient basis or on an agent by agent (or
combinations of agents). Thus, one can determine whether or not a
particular therapeutic treatment is likely to benefit a particular patient or
group/class of patients, or whether a particular treatment should be
continued.
The identified (IGEI) marker sets further provide previously unknown
or unrecognized targets for the development of anti-cancer agents, such as
CA 02820020 2013-06-26
43
chemotherapeutic compounds, and can be used as targets in developing
single agent treatment as well as combinations of agents for the treatment of
cancer.
EXAMPLES
A skilled artisan can readily recognize that there is no limit as to the
structural nature of the agents of the present invention. As such, without
further description, it is believed that one of ordinary skill in the art can,
using the preceding description and the following illustrative examples,
make and utilize the compounds of the present invention and practice the
claimed methods. The following working examples therefore, specifically
point out the preferred embodiments of the present invention, and are not to
be construed as limiting in any way the remainder of the disclosure.
In one embodiment, standardized RT (StaRT)-PCR, was employed to
assess various mutidrug resistant genes in a set of non-small cell lung
cancer (NSCLC) cell lines with a previously determined range of sensitivity
to cisplatin. Data were obtained in the form of target gene molecules relative
to 106 13-actin (ACTB) molecules. To cancel the effect of ACTB variation
among the different ells lines individual gene expression values were
incorporated into ratios of one gene to another. Each two-gene ratio was
compared as a single variable to chemoresistance for each of eight NSCLC
cell lines using multiple regression. Following validation, single variable
models best correlated with chemoresistance (p < 0.001 ), were determined.
In certain embodiments, the variable models included: ERCC2/XPC,
ABCC5/GTF2H2, ERCC2/GTF2H2, XPA/XPC and XRCC1/XPC All single
variable models were examined hierarchically to achieve two variable
models. The two-variable model with the highest correlation was
(ABCC5/GTF2H2, ERCC2/GTF2H2) with an R2 value of 0.96 (p < 0.001). In
certain embodiments, these markers are suitable for assessment of small
samples of tissue such as fine needle aspirate biopsies to prospectively
identify cisplatin resistant tumors.
StaRT-PCR is used to measure expression of 35 genes involved in
DNA repair, multi-drug resistance, cell cycling and apoptosis in two cell
lines
CA 02820020 2013-06-26
44
previously reported to be the least (H460) and most (H1435) chemoresistant
among 20 NSCLC cell lines. Weaver, D.A., Zahorchak, R., Varnavas, L.,
Crawford, EL., Warner, K.A., Willey, J.C, Comparison of expression
patterns by microarray and standardized RT-PCR analyses in lung cancer
cell lines with varied sensitivity to carboplatin, Proc Am Assoc Cancer Res
(2001 ) abstract, 42, 606. Tsai, CM., Chang, K.T., Wu, L.H., Chen, J.Y.,
Gazdar, A.F., Mitsudomi, T., Chen, M.H., Perng, R.P., Correlations between
intrinsic chemoresistance and HER-2/neu gene expression, p53 mutations,
and cell proliferation characteristics in non-small cell lung cancer cell
lines,
Cancer Res (1996), 56, 206-109. Genes involved in DNA repair (ERCC2,
XRCC1 ) and drug influx/efflux (ABCC5) are associated with
chemoresistance. The number of genes from each of these two categories
was expanded to include additional representative genes associated with
generalized DNA damage recognition and repair (DDIT3), associated
specifically with NER (LIG1, ERCC3, GTF2H2, XPA, XPC), or associated
with drug transport (ABCC1, ABCC4, ABCC10). Expression of these twelve
genes was measured in eight NSCCLC cell lines with variable cisplatin
resistance. Tsai, CM., Chang, K.T., Wu, L.H., Chen, J,Y., Gazdar, A.F.,
Mitsudomi, T., Chen, M.H., Perng, R.P., Correlations between intrinsic
chemoresistance and HER-2/neu gene expression, p53 mutations, and cell
proliferation characteristics in non-small cell lung cancer cell lines, Cancer
Res (1996), 56, 206-109. StaRT-PCR data were obtained using ACTB as a
reference gene. Thus, data were reported in the form of mRNA
molecules/106 ACTB molecules. These data then were combined into
interactive gene expression indices (IGEI) by placing one or more genes
directly associated with the phenotype on the numerator and one or more
genes negatively associated with the phenotype on the denominator using
the quantitative reverse transcriptase-PCR method described in the Willey
U.S. Patent Nos. 5,639,606; 5,643,765; and 5,876,978. Willey, J.C,
Crawford, E.L, Jackson, CM., Weaver, D.A., Hoban, J.C, Khuder, S.A.,
DeMuth, J.P.õ Expression measurement of many genes simultaneously by
quantitative RT-PCR using standardized mixtures of competitive templates,
Am J Respir Cell Mol Biol (1998), 19, 6-17. DeMuth, J.P., Jackson, CM.,
CA 02820020 2013-06-26
Weaver, D.A., Crawford, EL., Durzinsky, D.S., Durham, S.J., Zaher, A.,
Philips, ER., Khuder, S.A., Willey, J.C, The gene expression index c-myc x
E2F1/p21 is highly predictive of malignant phenotype in human bronchial
epithelial cells, Am J Respir Cell Mol Biol (1998), 19, 18-24. The IGEI are
5 geter predictors of phenotypes than are the expression levels of
individual
genes. For certain cancer-related phenotypes. Willey, J.C, Crawford, E.L.,
Jackson, CM., Weaver, D.A., Hoban, J.C, Khuder, S.A., DeMuth, J.P.õ
Expression measurement of many genes simultaneously by quantitative RT-
PCR using standardized mixtures of competitive templates, Am J Respir
10 Cell Mol Biol (1998), 19, 6-17. DeMuth, J.P., Jackson, CM., Weaver,
D.A.,
Crawford, E.L., Durzinsky, D.S., Durham, S.J., Zaher, A., Philips, E.R.,
Khuder, S.A., Willey, J.C, The gene expression index c-myc x E2F1/p21 is
highly predictive of malignant phenotype in human bronchial epithelial cells,
Am J Respir Cell Mol Biol (1998), 19, 18-24. Crawford, E.L., Khuder, S.A.,
15 Durham, S.J., Frampton, M., Utell, M., Thilly, VV.G., Weaver, D.A.,
Ferencak,
VV.J., Jennings, C.A., Hammersley, J.R., Olson, D.A., Willey, J.C, Normal
bronchial epithelial cell expression of glutathione transferase P1,
glutathione
transferase M3, and glutathione peroxidase is low in subjects with
bronchogenic carcinoma, Cancer Res (2000), 60, 1609-1618. Rots, J.G.,
20 Willey, J.C, Jansen, G., Van Zantwijk, CH., Noordhuis, P., DeMuth, J.P.,
Kuiper, E., Verrman, A.J., Pieters, R., Peters, G.J., mRNA expression levels
of methotrexate resistance-related proteins in childhood leukemia as
determined by a standardized competitive template-based RT-PCR method,
Leukemia (2000), 14, 2166-2175. A further advantage of IGEI is that they
25 control for previously observed variation in the reference gene value
(in this
case, ACTB) from one cell line to another. Willey, J.C, Crawford, E.L.,
Jackson, CM., Weaver, D.A., Hoban, J.C, Khuder, S.A., DeMuth, J.P.õ
Expression measurement of many genes simultaneously by quantitative RT-
PCR using standardized mixtures of competitive templates, Am J Respir
30 Cell Mol Biol (1998), 19, 6-17. DeMuth, J.P., Jackson, CM., Weaver,
D.A.,
Crawford, E.L, Durzinsky, D.S., Durham, S.J., Zaher, A., Philips, ER.,
Khuder, S.A., Willey, J.C, The gene expression index c-myc x E2F1/p21 is
highly predictive of malignant phenotype in human bronchial epithelial cells,
CA 02820020 2013-06-26
46
Am J Respir Cell Mol Blot (1998), 19, 18-24. When a single gene in the
numerator is divided by another single gene in the denominator, the
reference value mathematically cancels out. The IGEI values were
compared to cisplatin chemoresistance among the eight NSCLC cell lines
with variable resistance. Results then were validated in an additional six
NSCLC cell lines.
EXAMPLE I
Materials and Methods
Cell Culture
Non-small cell lung cancer (NSLC) cell lines H460, H1155, H23,
H838, H1334, H1437, H1355, H1435, H358, H322, H441, H522, H226 and
H647 were obtained from the American Type Culture Collection (Rockville,
MD). All cells were incubated in RPMI-1640 medium (Biofluids, Inc.,
Rockville, MD) containing 10% fetal bovine serum (FBS) and 1mM
glutamine at 37 C in the presence of 5% CO2. Proliferative, subconfluent
cultures were obtained from RNA extractions and subsequent analyses.
Reagents
10X PCR buffer for the RapidcyclerTM (500 mM Tris, pH 8.3; 2.5
mg/pi BSA, 30 mM MgC12) was obtained from Idaho Technology, Inc. (Idaho
Falls, ID). Taq polymerase (5 U/pl), oligo dT primers, RNasin (25 U/pl) and
dNTPs were obtained from Promega (Madison, WI). M-MLV reverse
transcriptase (200 U/pl) and 5X first strand buffer (250 mM Tris-HCI, pH 8.3;
375 mM KCI; 15 mM MgC12; 50 mM DTT) were obtained from Gibco BRL
(Gaithersburg, MD). DNA 7500 Assay kits containing dye, matrix and
standards were obtained from Agilent Technologies, Inc. (Palo Alto, CA). All
other chemicals and reagents were molecular biology grade.
RNA extraction and reverse transcription
Total RNA was isolated from cell cultures by a TriReagent protocol
(Molecular Research Center, Inc., Cincinnati, OH). Chomczynski, P., A
reagent for the single-step simultaneous isolation of RNA, DNA and proteins
from cell and tissue samples, Biotechniques (1993), 15, 536-537. Following
extraction, approximately 1 pg of total RNA for each cell line was reverse-
CA 02820020 2013-06-26
47
transcribed using M-MLV reverse-transcriptase and an oligo dT primer as
previously described in Willey, J.C, Coy, E., Brolly, C, Utell, M.J.,
Frampton,
M.W., Hammersley, J., Thilly, W.G., Olson, D., Cairns, K., Xenobiotic
metabolism enzyme gene expression in human bronchial epithelial and
alveolar macrophage cells, Am. J. Respir. Cell Biol. (1996), 14, 262-271.
Quantitative Standardized RT (StaRT)-PCR
Gene expression was determined using quantitative StaRT-PCR
protocols described in U.S. Patent Nos. 5,639,606; 5,643,765, and
5,876,978 and in Willey, J.C, Crawford, E.L., Jackson, CM., Weaver, D.A.,
Hoban, J.C, Khuder, S.A., DeMuth, J.P., Expression measurement of many
genes simultaneously by quantitative RT-PCR using standardized mixtures
of competitive templates, Am J Respir Cell Mol Biol (1998), 19, 6-17. Willey,
J.C, Coy, E., Brolly, C, Utell, M.J., Frampton, M.W., Hammersley, J., Thilly,
W.G., Olson, D., Cairns, K., Xenobiotic metabolism enzyme gene
expression in human bronchial epithelial and alveolar macrophage cells, Am
J Respir Cell Biol (1996), 14, 262-271. Apostolakos, M.J., Schuermann,
W.H., Frampton, M.W., Utell, M.J., Willey, J.C, Measurement of gene
expression by multiplex competitive polymerase chain reaction, Anal.
Biochem. (1993), 213, 277-284. Willey, J.C, Coy, E.L. Frampton, M.W.,
Torres, A., Apostolakos, M.J., Hoehn G., Schuermann, W.H. Thilly W.G.,
Olson, D.E., Hammersley, J.R., Crepsi, C.L. Utell, M.J., Quantitative RI-
PCR measurement of cytochromes p4a50 1A1, 1131, and 2B7, microsomal
epoxide hydrolase, and NADPH oxidereductase expression in lung cells of
smokers and non-smokers. Am. J. Respir. Cell Mol. Biol. (1997) 17, 114-
124. Briefly, a master mixture containing buffer, MgC121 dNTPs, sample
cDNA, Taq polymerase and competitive template (CT) mixture was
prepared and 9 pl aliquots dispensed into 0.6 ml microfuge tubes containing
1 pl of gene-specific primers. The CT mixture comprises gene-specific
internal standard competitive templates (CTs) at defined concentrations
relative to one another and also contains CT for a housekeeping gene,
ACTB, to allow for the normalization of all specific gene data. All primers
used for PCT and those used in the construction of the CTs, are listed in
Table 1. PCR reactions were subjected to 35 cycles of PCR with 5 seconds
CA 02820020 2013-06-26
48
of denaturation at 94 C, 10 seconds of annealing at 58 C and 15 seconds of
elongation at 72 C in a Rapidcycler (Idaho Technology, Inc.). FOR products
were electrophoretically separated and quantified in an Agilent 2100
Bioanalyzer (Agilent Technologies, Inc.) with the DNA 7500 Assay Kit.
Chemoresistance of NSCLC cell lines
Chemoresistance IC50 (pm) values of the NSCLC cell lines used for
several chemotherapeutic agents were previously determined, as described
in Tsai, CM., Chang, K.T., Wu, L.H., Chen, J.Y., Gazdar, A.F., Mitsudomi,
T., Chen, M.N., Perng, R.P., Correlations between intrinsic chemoresistance
and HER-2/neu gene expression, p53 mutations, and cell proliferation
characteristics in non-small cell lung cancer cell lines, Cancer Res (1996),
56, 206-109 and are summarized for cisplatin in Table 2.
Statistical Analyses
Ratios of one gene to another, from each of the initial eight NSCLC
cell lines, were subjected to multiple regression analysis with SAS (version
6, 4th edition, volume 2) statistical package (SAS Institute Inc., Cary, NC)
to
determine the combination of genes that best predict cisplatin resistance.
Each ratio was compared separately to chemoresistance and ratios with
significant correlation to resistance (R2 > 0.88, p < 0.001 ) then were
examined hierarchically to achieve two variable models based on the
highest R2 values. Following assessment of an additional 6 cell lines, results
for all 14 NSCLC cell lines were combined and subjected to analysis as
described.
RESULTS: Reproducibility
Among the gene expression measurements for which three or more
replicate values were obtained, the mean coefficient of variation was 38.5%
(raw data available at website). This is similar to the reproducibility
observed
in other gene expression studies using the StaRT-FOR method. Willey, J.C,
Crawford, EL, Jackson, CM., Weaver, D.A., Hoban, J.C, Khuder, S.A.,
DeMuth, J.P., Expression measurement of many genes simultaneously by
quantitative RT-PCR using standardized mixtures of competitive templates,
Am. J. Respir. Cell Mol. Biol. (1998), 19, 6-17. Crawford, E.L, Khuder, S.A.,
Durham, S.J., Frampton, M., Utell, M., Thilly, W.G., Weaver, D.A., Ferencak,
CA 02820020 2013-06-26
49
W.J., Jennings, C.A., Hammersley, J.R., Olson, DA, Willey, JO, Normal
bronchial epithelial cell expression of glutathione transferase P1,
glutathione
transferase M3, and glutathione peroxidase is low in subjects with
bronchogenic carcinoma, Cancer Res. (2000), 60, 1609-1618.
Individual Gene Expression Measurements and Chemoresistance
The results of the direct comparison of individual gene expression
mean values versus cisplatin chemoresistance for the first set of eight cell
lines (Group 1) are presented in Table 3. All StaRT-PCR data values were
in the form of molecules/106 ACTB molecules. For 8/12 genes assessed,
there was significant (p<0.05) correlation.
Establishment of inter-active gene expression ratios
IGEI were established comprising every possible combination of the
expression value of one gene divided by the expression value of another
gene for data obtained from each of the initial eight NSCLC cell lines (Group
1 ). Each expression value was calculated as molecules/106 ACTB
molecules. Thus, in these IGEI the effect of the reference gene, ACTB, is
cancelled. For Example:
ERCC2 molecules/106 ACTB molecules XPC molecules/106
ACTB molecules = ERCC2 molecules/XPC molecules.
Bivariate analysis of each two-gene ratio versus corresponding
cisplatin 1050 chemoresistance values was conducted among the eight cell
lines (Table 4). There were 12 genes assessed and 11 sets of ratios for
each gene resulting in 132 ratios. The sets of 11 ratios for each gene then
were organized in descending order such that the ratio set listed first was
that for which the average correlation with chemoresistance was highest,
and the ratio set listed last was that for which the average correlation with
chemoresistance was lowest. Thus the ratio set with ERCC2 in the
numerator is listed first because the average of the r values for the ratios
between ERCC2 and each of the other eleven genes was the most positive
among the twelve genes evaluated. In contrast, the ratio set with XPC in the
numerator is listed last because the ratios between XPC and each of the
other 11 genes had the most negative correlation with chemoresistance.
CA 02820020 2013-06-26
Modeling of gene expression with chemoresistance
The ratios ERCC2/XPC, ABCC5/GTF2H2, ERCC2/XRCC1,
ERCC2/GTF2H2, XPA/XPC, XRCC1/XPC, and ABCC5/XPC were the best
single variable models (i.e., those with R2 > 0.87) identified in the initial
eight
5 NSCLC cell lines by simple linear regression (Table 5). The effect of
adding
a second variable into the model was then assessed. The best two variable
model was (ABCC5/GTF2H2, ERCC2/GTF2H2) with an R2 value of 0.96.
Validation of Models
These single and two variable models were tested in an additional six
10 NSCLC cell lines. From the statistical analysis of the combined data for
all
14 NSCLC cell lines, the p value improved or stayed the same for three of
the single variable models (ERCC2/XPC, ABCC5/GTF2H2, XRCC1/XPC),
as well as the two variable model. The decline in p value for
ERCC2/GTF2H2 and XPA/XPC was small and not significant. In contrast,
15 ERCC2/XRCC1 was no longer significantly associated with
chemoresistance, and the p value declined substantially for ABCC5/XPC
Analysis of Results
The results obtained by measuring gene expression with StaRT-PCR,
incorporating values for individual genes into IGEI, and correlating IGEI with
20 chemoresistance provides several models useful as predictors of
cisplatin
chemoresistance in cultured NSCLC cells. These models comprise genes
associated with cisplatin chemoresistance, including ABCC5, ERCC2, XPA,
and XRCC1. Increased expression of ABCC5, also known as MRP5, is
associated with exposure to platinum drugs in lung cancer in vivo and/or the
25 chronic stress response to xenobiotics. Thus, increased resistance to
platinum drugs with increased ABCC5 levels may be due to glutathione S-
platinum complex efflux.
The remaining genes directly associated with chemoresistance, XPA
and ERCC2, are components of the nucleotide excision repair (NER)
30 mechanism which generally is recognized as the major repair response to
DNA damage induced by chemotherapeutic agents such as cisplatin. In
NER, XPA is the main DNA lesion recognition protein (Asahina, H.,
Kuraoka, I., Shirakawa, M., Morita, E.H., Miura, N., Miyamoto, I., Ohtsuka,
CA 02820020 2013-06-26
51.
E., Okada, Y., Tanaka, K., The XPA protein is a zinc metal loprotein with an
ability to recognize various kinds of DNA damage, Mutat. Res. DNA Repair
(1994), 315, 229-237) and is the key element in assembly of the NER
complex by recruiting several other proteins to the lesion site. Li, L.
Peterson, C.A., Lu, X., Legerski, R.J., Mutations in XPA that prevent
association with ERCC1 are defective in nucleotide excision repair, Mol Cell
Biol (1995), 15, 1993-1998. Enhanced NER gene expression has been
shown to be a major cause of resistance to cisplatin and other DNA-
damaging chemotherapeutic agents (Zamble, D.B., Lippard, S.J., Cisplatin
and DNA repair in cancer chemotherapy, Trends Biochenn. Sci. (1995), 20,
435-439, Reed, E., Anticancer drugs: platinum analogs. In: Cancer:
Principles and Practice of Oncology, (1993), 390-399. Editors V.T. Devita,
Jr., S. Hellman and S.A. Rosenberg, Lippincott, Philadelphia) and
overexpression of the XPA gene component of NER has been associated
with resistance to cisplatin in human ovarian cancer. Dabholkar, M., Vionnet,
J., Bostick-Bruton, F., Yu, J.J., Reed, E., Messenger RNA levels of XPAC
and ERCC1 in ovarian cancer tissue correlate with response to platinum-
based chemotherapy, J. Olin. Invest. (1994), 94, 703-708. ERCC2
specifically is a component of the transcription factor IIH (TFIIH) which
consists of seven polypeptides (Mu, D., Park, OH., Matsunaga, T., Hsu,
D.S., Reardon, J.T., Sancar, A., Reconstitution of human DNA repair
excision nuclease in a highly defined system, J. Biol. Chem. (1995), 270,
2415-2418, Mu, D., Hus, D.S., Sancar, A., Reaction mechanism of human
DNA repair excision nuclease, J. Biol. Chem. (1996), 271,8285-8294) and in
its entirety is a repair factor. Schaeffer, L., MoncoIlin, V., Roy, R., Staub,
A.,
Mezzina, M., Sarasin, A., Weeda, G., Hoeijmakers, J.H., Egly, J.M., The
ERCC2/DNA repair protein is associated with the class ll BTF2/TFIIH
transcription factor, EMBO J (1994), 13, 2388-2392, Drapkin, R., Reardon,
J.T., Ansari, A, Huang, JO, Zawel, L, Ahn, K., Sancar, A., Reinberg, D.,
Dual role of TFIIH in DNA excision repair and in transcription by RNA
polymerase II, Nature (1994), 368, 769-772, Wang, Z., Svejstrup, J.Q.,
Feaver, W.J., Wu, X., Kornberg, R.D., Friedberg, E.C, Transcription factor b
(TFIIH) is required during nucleotide-excision repair in yeast, Nature (1994),
CA 02820020 2013-06-26
52
368, 74-76. In NER, ERCC2 (or XPD) is essential for TFIIH helicase activity
(Prakash, S., Sung, P., Prakash, L., DNA repair genes and proteins of
Saccharoyces cerevisiae, Annu. Rev. Genet. (1993), 27, 33-70), and it has
been demonstrated more recently that ERCC2 interacts specifically with
GTF2H2 (or p44) and this interaction results in the stimulation of the 5' to
3'
helicase activity. Coin, F., Marinoni, J.C, Rodoflo, C, Fribourg, S.,
Pedrinin,
A.M., Egly, J.M., Mutations in the XPD helicase gene result in XP and TTD
phenotypes, preventing interaction between XPD and the p44 subunit of
TFIIH, Nature Genet, 20, 184-188.
With microarray analysis, because thousands of genes are assessed
simultaneously, an index of all genes measured provides a stable reference
for the amount of sample loaded from one nnicroarray to another. In
quantitative RT-PCR studies, typically, a single non-regulated gene is used
as a loading reference, such as ACTB, GAPDH, cyclophilin or ribosomal
RNA. However, all of these genes have been reported to vary among
multiple samples. One way to assess inter-sample variation in reference
gene expression among multiple samples is to compare variation between
two reference genes, 6-actin and GAPDH vary 50-fold relative to each other
among bronchial epithelial cells (BEC) and even more between BEC and
other cell types. Willey, J.C, Crawford, E.L., Jackson, CM., Weaver, D.A.,
Hoban, J.C, Khuder, S.A., DeMuth, J.P., Expression measurement of many
genes simultaneously by quantitative RT-PCR using standardized mixtures
of competitive templates, Am. J. Respir. Cell Mol Biol. (1998), 19, 6-17.
Rots, J.G., Willey, J.C, Jansen, G., Van Zantwijk, C.H., Noordhuis, P.,
DeMuth, J.P., Kuiper, E., Verrman, A.J., Pieters, R., Peters, G.J., mRNA
expression levels of methotrexate resistance-related proteins in childhood
leukemia as determined by a standardized competitive template-based RT-
PCR method, Leukemia (2000), 14, 2166-2175. In situations where limited
numbers of genes are measured (< 200), an index of all genes for the
normalization of data is not sufficiently stable. In order to eliminate the
effect
of unknown variation in the reference gene expression among samples,
balanced ratios of one gene expression value obtained by StaRT-PCR to
another were analyzed. These balanced ratios did not represent actual
CA 02820020 2013-06-26
53
cellular concentration changes of the individual genes comprising the ratio,
but related the expression of gene to another and are used for comparison
with phenotypic determinants such as chemoresistance. In this study, IGEI
analysis (Table 5) confirmed most of the results obtained by analysis of
individual gene expression values relative to chemoresistance (Table 3).
Specifically, XPC was the most stable of the twelve genes assessed relative
to chemoresistance and the same eight genes were correlated with
chemoresistance using XPC as the denominator (Table 4) as was the case
using 13-actin as the denominator (Table 3). Thus, variation in 3-actin among
this group of cDNA samples was not significant. In certain embodiments, it is
useful to use IGEI to remove doubt regarding potential effect of variation in
reference gene expression whenever possible.
As is presented in Table 4, by evaluating an empirically derived set of
balanced ratios (IGEI) derived from expression values for all of the genes
measured, it is possible to establish a hierarchy regarding the strength of
association between a set of genes and a phenotype. Further, bivariate
correlation of each gene relative to each of the others markedly increases
the power of the analysis and helps to identify potential outliers that
require
further validation. In the example herein, the most obvious outlier is the
high
correlation between ERCC2/XRCC1 and chemoresistance. This is an outlier
because (a) the sets of ratios with ERCC2 or XRCC1 in the numerator had
the highest and fourth highest range r values respectively (Table 4), yet (b)
all of the other ratios with ERCC2 in the numerator that had high r values
had genes from the bottom of Table 4 in the denominator (i.e. XPC,
GTF2H2, ABCC10, ERCC3, and Lig1 all were among the lowest in the
table). Consistent with the evidence that ERCC2/XRCC1 is an outlier, when
the Group 2 cell lines were evaluated, ERCC2/XRCC1 was no longer
significantly associated with chemoresistance (Table 5). These findings
provide further evidence for the value of measuring gene expression in
standard, numerical format.
Thus, the association of ERCC2, ABCC5, XPA, and XRCC1 with
chemoresistance is established through a sequential process involving (a) a
first round of screening genes representing many different functional
CA 02820020 2013-06-26
54
classes, (b) evaluating an expanded group of genes represented by those
that are positively associated in the first round, (c) combining the
positively
connected data into interactive gene expression indices (IGEI), (d) using
IGEI analysis to identify outliers, (e) building a model and (f) validating
the
data.
The method of the present invention highlights the necessity to
evaluate the interaction of more than one gene involved in cisplatin
chemoresistance and the interaction of multiple pathways that may give rise
to chemoresistance.
EXAMPLE II
The identification of many genes and their association to specific
phenotypes will most likely lead to molecular cancer classification (Venter,
J.C. The sequence of the human genome, Science, 291 :1304-1351 (2001 ),
Lander, E.S., Initial sequencing and analysis of the human genome, Nature,
409:860-921 (2001 ). This novel classification system has important clinical
implications and may greatly improve patient care. Specifically, recognition
of certain genotypes with associated phenotypes may reveal individual
prognostic markers, chemosensitivity traits, and predict patient outcome.
Molecular classification of lung cancer may greatly enhance cytologic
diagnosis. Lung cancer is still primarily diagnosed using histopathological
criteria. The heterogeneity of lung tumors often leads to inconsistent
diagnosis (Sorenson, J.B., Hirsch, FR., Gazdar, A., and Olsen, J.E.,
Cancer, 71 :2971-2976, 1993), including difficulty distinguishing malignant
from normal and metastatic lung tumors from primary tumors (Shirakusa, T.,
Tsutsui, M., Motomaga, R. Ando, K. and Kusano T., A. Surg., 54:655-658,
1966; Fling, A. and Lloyd, R.V., Arch. Pathol. Lab. Med. 166: 39-42, 1992).
Gene expression patterns have clarified clinical outcomes in lung and
breast cancer patients. Garber et al., Proc. Natl. Acad. Sci. 98: 13874-
113789 (2001 ) reported gene expression profiles of lung tumors correlated
with transitional morphological classification. In addition, based on gene
expression patterns, adenocarcinomas were further divided into 3 subtypes
that differed significantly in patient survival. Bhattacharjee et al., Proc.
Natl.
CA 02820020 2013-06-26
Acad. Sci., 98: 13799990-13795 (2001) reported similar results. Lung
adenocarcinomas were grouped into 4 subclasses based on gene
expression patterns, and patients had statistically significant differences in
survival. They also identified three metastatic lung tumors based on gene
5 expression
profile that were morphologically identified as primary lung
tumors. Molecular classification of breast cancer tumors based on gene
expression profiles and correlation to patient outcome and cell proliferation
rates have also been reported in cases of hereditary breast cancer, sporadic
breast cancer and human mannary epithelial cells (Hendenfalk, et al., New
10 Eng. J. of
Med. 344: 539-548, 2001; Sorlie et al., Proc. Natl. Acad. Sci. 98:
10869-10874, 2001; and Perou et al., Distinctive gene expression patterns
in human mammary epithelial cells and breast cancers Proc. Natl. Acad. Sci.
USA vol.96, no. 16: 9212-9217,1999).
Most lung cancers are diagnosed primarily by fine-needle aspirate
15 (FNA) biopsy
tissues, pleural fluid samples and brushings of bronchial
epithelial cells. These small, non-renewable tissue samples are challenging
to use in gene expression studies. Microarray methods are appropriate for
screening thousands of genes potentially involved in numerous cancer
phenotypes, however they are unsuitable for FNA gene expression analysis
20 because of
large initial RNA amounts required, lack of internal standards,
cost and time (Tyagi, S. and Kramer, F.R., Nature Biotech. 14: 303-308,
1996; DeRisi, J.L., Science, 278: 6860-6866, 1997). After target gene
identification, gene expression analysis should be further evaluated with a
quantitative, standardized gene expression method.
25 StaRT-PCR
(Standardized Reverse Transcriptase-Polymerase Chain
Reaction) is an ideal gene expression method to use in small clinical
samples. It is useful to measure hundreds of genes simultaneously, requires
small amounts of RNA, uses inexpensive equipment, is sensitive,
standardized and highly reproducible (Willey et al., AM. J. Respir. Cell Mol.
30 Biol. 19: 6-
17, 1998, Crawford et al., Crawford, E.L., Godfridus, J. Peters,
Noordhuis, P., Rots, M.G., Vondracek, M., Grafstrom, R.C, Lieuallen, K.,
Lennon, G., Zahorchak, R.J., Georgeson, M.J., Wali, A., Lechner, J.F., Fan,
P-S., Kahaleh, B., Khuder, S.A., Warner, K.A., Weaver, D.A., and Willey,
CA 02820020 2013-06-26
56
J.C. (2001 ), Reproducible gene expression measurement among multiple
laboratories obtained in a blinded study using standardized RT (StaRT)-
PCR, Molecular Diagnosis 6: 217-225, 2001). It is likely that malignant,
chemoresistant and metastatic phenotypes result from the interactive effects
of many genes. Because the data are numerical in StaRT-PCR studies,
phenotypes can be represented by interactive gene expression indicies
GED. Demuth et al., Am. J. Respir. Cell Mol. Biol. 19: 18-24, 1998,
reported the gene expression index of c-myc x E2F-1/p21 predicted
malignancy in human bronchial epithelial cells better than any individual
gene measured. In a similar study, the gene expression index of mGST x
GSTM3 x GSHPx x GSHPxA x GSTP1 was sensitive (90%) and 76%
specific for detecting normal bronchogenic epithelial cells from subjects with
bronchogenic carcinoma (Crawford et al., Cancer Research, 60: 1609-1618,
2000). Specifically, this interactive gene expression index identified
individuals at risk for developing bronchogenic carcinoma better than any
single gene.
The inclusion of standardized, competitive templates in every StaRT-
PCR reaction allows direct intra-laboratory and inter-laboratory data
comparison (Willey et al., 1998). Crawford et al., (2001) reported high inter-
laboratory reproducibility using StaRT-PCR. (Crawford, E.L., Godfridus, J.
Peters, Noordhuis, P., Rots, M.G., Vondracek, M., Grafstrom, R.C,
Lieuallen, K., Lennon, G., Zahorchak, R.J., Georgeson, M.J., Wali, A.,
Lechner, J.F., Fan, P-S., Kahaleh, B., Khuder, S.A., Warner, K.A., Weaver,
D.A., and Willey, J.C. (2001 ), Reproducible gene expression measurement
among multiple laboratories obtained in a blinded study using standardized
RT (StaRT)-PCR, Molecular Diagnosis 6:217-225, 2001 ). The generation of
standardized, numerical data is needed for establishing a common, multi-
institutional database. A recent modification of StaRT-PCR, termed multiplex
standardized RT-PCR, allows further reduction in the amount of starting
material needed for gene expression studies (Crawford, EL., Warner, K.A.,
Khuder, S.A., Zahorchak, R.J., and Willey, J.C, Multiplex standardized RT-
PCR for expression analysis of many genes in small clinical samples,
Biochemical and biophysical Research Communications, 293: 509-516,
CA 02820020 2013-06-26
57
2002). Using multiplex StaRT-PCR at least 96 may be simultaneously
evaluate using the same amount of cDNA that is normally used for
measurement of one gene. (Crawford, et al. 2002, supra). This method was
used to simultaneously measure 18 genes putatively associated with
chemoresistance in a bronchogenic carcinoma sample obtained by FNA.
This example determines if a high c-myc x E2F-1/p21 gene
expression index could augment cytopathological diagnosis of bronchogenic
carcinoma. Standardized gene expression values for c-nnyc, E2F-1 and p21
and the interactive gene malignancy index were determined for eight
primary lung FNA samples.
Materials and Methods
Cell Culture
The H1155 human NSCLC cell line was purchased from ATCC
(Manassas, VA), and cultured (37 C, 5.0% CO2) in RPMI supplemented
with gentamicin (0.1 %) (Biofluids, Rockville, MD) and 10% fetal bovine
serum (FBS) (Sigma, St. Louis, MO).
Evaluation of RNA Preservation, Extraction and Reverse Transcription
H1155 cells (1.0 E6) were placed in Preservcyt (CYTYC/Boxborough,
MA), RNA-Later (Ambion/Austin, Texas) or Tr-Reagent (Molecular
Research Center, Cincinnati, OH) prior to RNA extraction. Time points and
temperatures evaluated for RNA quality were 1, 3, 10 and 30 days and room
temperature, 4 C and -20 C RNA was extracted from cells using Tri
Reagent according to manufacturer's protocol. After extraction, RNA quality
was evaluated on an Agilent 2100 Bioanalyzer for detection of 18s and 28s
ribosomal peaks. MRNA samples were reverse transcribed using M-MLV
reverse transcriptase (Gibco BRL, Gaithersburg, MD) and oligo (dT) primer
(Promega, Madison, WI) as previously described. (DeMuth, J.P., Jackson,
CM., Weaver, D.A., Crawford, E.L, Durzinsky, D.S., Durham, S.J., Zaher, A.,
Phillips, E.R., Khuder, S.A. and Willey, J.C. (1998), The gene expression
index of c-myc x E2F-1/p21 is highly predictive of malignant phenotype in
human bronchial epithelial cells, Am. J. Respir. Cell Mol. Biol., 19, 18-24.
Crawford, E.L., Khuder, S.A., Durham, S.J., Frampton, M., Utell, M., Thilly,
VV.G., Waver, D.A., Ferencak, VV.J., Jennings, C.A., Hammersley, J.R.,
CA 02820020 2013-06-26
58
Olson, D.A., and Willey, J.C. (2000), Normal bronchial epithelial cell
expression of the glutathione transferase P1, Glutathione transferase M3,
and Glutathione peroxidase is low in subjects with bronchogenic carcinoma,
Cancer Research, 60, 1609-1618.)
Uniplex-StaRT-PCR
StaRT-PCR was performed using previously published protocols
(Willey, J.C, Crawford, E.L., Jackson, CM., Weaver, D.A., Hoban, J.C,
Khuder, S.A., DeMuth, J.P. (1998), Expression measurement of many
genes simultaneously by quantitative RT-PCR using standardized mixtures
of competitive templates, Am. J. Respir. Cell Mol. Biol., 19, 6-17. DeMuth,
J.P., Jackson, CM., Weaver, D.A., Crawford, E.L., Durzinsky, D.S., Durham,
S.J., Zaher, A., Phillips, E.R., Khuder, S.A., Willey, J.C, (1998), The gene
expression index of c-myc x E2F-1/p21 is highly predictive of malignant
phenotype in human bronchial epithelial cells, Am. J. Respir. Cell Mol. Biol.,
19, 18-24. Crawford, E.L., Khuder, S.A., Durham, S.J., Frampton, M., Utell,
M., Thilly, W.G., Weaver, DA, Ferencak, W.J., Jennings, CA, Hammersley,
J.R., Olson, DA, Willey, J.C, (2000), Normal bronchial epithelial cell
expression of the glutathione transferase P1, Glutathione transferse M3, and
Glutathione peroxidase is low in subjects with bronchogenic carcinoma,
Cancer Research, 60, 1609-1618. Crawford, E.L, Godfridus, J.P.,
Noordhuis, P., Rots, M.G., Vondracek, M., Grafstrom, R.C, Lieuallen, K.,
Lennon, G., Zahorchak, R.J., Georgeson, M.J., Wali, A., Lechner, J.F., Fan,
P-S., Kahaleh, B., Khuder, S.A., Warner, K.A., Weaver, DA., Willey, J.C,
(2001), Reproducible gene expression measurement among multiple
laboratories obtained in a blinded study using standardized RT (StaRT)-
PCR, submitted. Gene Express System 1 Instruction Manual, Gene Express
National Enterprises, Inc. (2000), with G.E.N.E. system I expression kit
(Gene Express National Enterprises, Inc.).
There were six CT mixtures A-F and appropriate primers included in
System 1 kit. The concentration of "target gene" CTs varies in each mix
compared to the concentration of the "reference gene" actin. The master mix
contained Rnase-free water, MgC12 buffer, dNTPs, cDNA, CT mixture from
G.E.N.E. system I kit and taq polymerase. The master mix was placed into
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59
tubes containing individual gene primers, and cycled in a Rapidcycler (Idaho
Technology, Inc., Idaho Falls, ID). The denaturing temperature was 94 C,
annealing temperature was 58 C and elongation temperature was 72 C for
each cycle. After amplification, each per product was analyzed by capillary
electrophoresis on an Agilent 2100 Bioanalyzer machine. The area under
the curve of each native template (NT) was compared to that of its
respected competitive template (CT) to determined gene expression values.
The unit for each expression value was molecules per 106 13-actin
molecules.
Acquisition of Bronchogenic Carcinoma Samples
Fine needle aspirate (FNA) of primary lung cancer were obtained
from patients at the Medical College of Ohio. An informed, signed consent
was obtained from patients according to NIH and institutional guidelines
prior to each procedure. Most cells were placed directly on slides for
diagnostic purposes. Cells not needed for diagnostic purposes were
collected in Preservcyt Solution (CYTYC/Boxborough, MA). After final
cytopathologic diagnosis, remaining cells in Preservcyt were pelleted in our
laboratory and RNA was extracted. Cell number and viability were evaluated
on cells through analysis of cells on glass slides.
Results
In an effort to determine optimal collection and preservation of RNA in
FNA specimens, H1155 cells (NSCLC) were placed in 3 storage reagents,
RNA Later, Preservcyt and Tri Reagent. To determine effects of time and
temperature on RNA, H1155 cells were kept at 4 C or -20 C for I, 3, 10 and
30 days.
High quality RNA, indicated as ++ (exhibited the presence of 18s and
28s ribosomal bands) was detected in H1155 NSCLC cells stored in each
reagent up to 10 days (Table 6). RNA was preserved equally well in
Preservcyt and TRI reagent after 30 day storage. RNA was partially
degraded after 30 day storage in RNA Later (+-) at 4 C and was not
preserved at -20 C
To determine if RNA was suitable for StaRT-PCR, it was reverse
transcribed and 13-actin expression was evaluated, 13-actin was detected in
CA 02820020 2013-06-26
all samples exhibiting high quality or partially degraded RNA (Fig 6 -Table
6). As expected, (3-actin was not detected in cells stored in RNA later for 30
days at -20 C RNA quality correlated highly with the ability to be per
amplied. Optimal storage reagents for short term storage (1-10 days) are
5 Preservcyt and RNA later and for long term storage, greater than 10 days
Preservcyt is recommended. Preservcyt is also advantageous to use at
institutions that utilize the Thin Prep System for cytological analysis.
After determination of optimal collection and storage conditions, lung
FNA specimens were placed in Preservcyt and stored at 4 C Similar to the
io H1155 cells, RNA quality was evaluated in 9 of 10 FNA specimens (Fig 7 -
Table 7). Five samples had high quality (++) or partially degrated (+-) RNA.
As expected, all five samples were per amplifiable and 13-actin was detected.
On sample was not evaluated (NE) prior to reverse transcription and 4
samples exhibited poor quality RNA(¨ ). 13-actin was detected in the NE
15 specimen and unexpectedly was detected in 2 of the RNA (--) samples.
When high quality RNA is present, it is highly suitable for PCR experiments.
When poor quality RNA is present, it less likely to be per amplifiable but
still
may be useful.
In an attempt to determine why 4 samples had poor quality RNA, the
20 cytological characteristics were determined independently by a pathologist
for each specimen. Cellularity, viability and percent tumor/normal cells wre
determined for each sample (Table 7). Seven of 10 samples had low
cellularity (L) and low viability (L). Three of these samples had a (++) or (+-
)
RNA status and all were per amplifiable (+13-actin). Of the remaining four
25 samples with low cellularity and low viability, two were pramplifiable
and two
were not. Three of 10 samples had intermediate (I) or high (H) cellularity and
viability. All three had good quality RNA and were per amplifiable. It is
likely
that cellularity is related to the amount of RNA extracted and viability may
be
related RNA quality obtained from these cells. Specimens and intermediate
30 to high cellularity are optimal for gene expression studies, but cells
with low
cellularity and low viability are still suitable, since 5 of 7 were per
amplifiable.
In 7 of 10 samples, the % of tumor cells varied from 60-90%. Two
samples had 20% tumor cells and one was 10% tumor cells. The FNA
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61
diagnosis, determined at time of same acquisition was NSCLC in 6 samples
and atypical in 4. To confirm the presence of a malignant phenotype, 3
genes associated with malignancy, c-myc, E2F-1 and p21 were evaluated in
8 of 10 FNA's and the malignancy index of c-myc x E2F-1/p21 was
determined (Fig. 8 - Table 8). As expected, 5 of the +NSCLC samples had a
very high index value that ranged from 1.0E4 to 3.6E6 (as molecules per 106
13-actin molecules). Three of the four atypical samples also exhibited high
malignant gene expression indices, with values ranging from 7.2E3-5.0E4.
After additional analysis, the three atypical samples that gene expression
data was obtained from were later confirmed as small cell lung cancer
(SCLC). The percentage of tumor cells in the atypical samples ranged from
to 80% indicating even a small number of abnormal cells were sufficient
and detected by the gene expression index c-myc x E2F-1/p21.
While, FNA analysis of pulmonary nodules is a common diagnostic
15 method, this is the first example to use a standardized, quantitative
gene
expression method on human lung FNA samples. Gene expression profiling
of these small, non-renewable cell populations have diagnostic and
prognostic implications and lead to individualized patient care. Different
gene expression patterns are useful to discriminate between SCLC and
20 NSCLC, and earlier identification of a malignant phenotype will optimize
clinical treatment. In addition, StaRT-PCR is also useful to identify gene
expression patterns and associate them with clinically relevant phenotypes,
e.g. chemosensitivity and metastatic potential to improve patient prognosis.
In this example, 5 of the FNA samples initially diagnosed as NSCLC,
and later confirmed to be NSCLC had high index values. The range of
expression for these +NSCLC specimens were 1.0E4-3.6E6. In this sample
set, 4 were 90.0% tumor cells and sample #172 had only 20% tumor cells,
yet had the highest index value, 6.5E5. Three of the FNA samples,
cytologically diagnosed as atypical, and later confirmed to be SCLC also
had high index values. They ranged from 7.20E3-5.0E4 mRNA molecules
per 106 molecules 13-actin mRNA. The percentage of tumor cells in these
samples ranged from 20-80%.
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OTHER EMBODIMENTS
The genes and IGEI marker sets described herein provide valuable
information for the identification of new drug targets against NSCLC, and
that information may be extended for use in the study of carcinogenesis in
other tissues. These sequences may be used in the methods of the
invention or may be used to produce the probes and arrays of the invention.
The present invention is not to be limited in scope by the specific
embodiments described herein, but are intended as single illustrations of
individual aspects of the invention and it is to be understood that
functionally
equivalent methods and components are within the scope of the invention,
in addition to those shown and described herein and will become apparent
to those skilled in the art from the foregoing description and accompanying
drawings. Such modifications are intended to fall within the scope of the
appended claims.
