U.S. patent application number 10/336659 was filed with the patent office on 2004-04-15 for methods for assessing efficacy of chemotherapeutic agents.
Invention is credited to Kornblith, Paul L., McDonald, Sean.
Application Number | 20040072722 10/336659 |
Document ID | / |
Family ID | 32072939 |
Filed Date | 2004-04-15 |
United States Patent
Application |
20040072722 |
Kind Code |
A1 |
Kornblith, Paul L. ; et
al. |
April 15, 2004 |
Methods for assessing efficacy of chemotherapeutic agents
Abstract
Methods are provided for accurately predicting efficacy of
chemotherapeutic agents. Methods of the invention increase the
positive predictive value of chemosensitivity assays by assessing
both the ability of a chemotherapeutic to destroy cells and the
genetic propensity of those cells for resistance. Results obtained
using methods of the invention provide insight into the in vivo
effectiveness of a therapeutic, and lead to more effective
chemotherapeutic treatment.
Inventors: |
Kornblith, Paul L.;
(Pittsburgh, PA) ; McDonald, Sean; (Pittsburgh,
PA) |
Correspondence
Address: |
TESTA, HURWITZ & THIBEAULT, LLP
HIGH STREET TOWER
125 HIGH STREET
BOSTON
MA
02110
US
|
Family ID: |
32072939 |
Appl. No.: |
10/336659 |
Filed: |
January 2, 2003 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
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60417439 |
Oct 10, 2002 |
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Current U.S.
Class: |
514/1 ;
435/6.16 |
Current CPC
Class: |
C12Q 1/6886 20130101;
C12Q 1/6883 20130101; C12Q 2600/106 20130101; C12Q 2600/136
20130101 |
Class at
Publication: |
514/001 ;
435/006 |
International
Class: |
A61K 031/00; C12Q
001/68 |
Claims
What is claimed is:
1. A method for assessing efficacy of a chemotherapeutic agent, the
method comprising: conducting an assay to determine whether a
chemotherapeutic agent affects cellular phenotype of a sample of
tumor cells obtained from a patient; determining whether said
patient comprises a genetic characteristic associated with
resistance to said chemotherapeutic agent; and assessing efficacy
of said chemotherapeutic agent based upon results of said
conducting and detecting steps.
2. The method of claim 1, wherein said sample of cells in said
conducting step comprise malignant cells.
3. The method of claim 1, wherein said sample of cells in said
conducting step comprise abnormal proliferating cells.
4. The method of claim 1, wherein said cellular phenotype is cell
growth rate or death.
5. The method of claim 1, wherein said genotypic change is a
genetic polymorphism or mutation.
6. The method of claim 1, wherein said determining step comprises
sequencing a portion of the genome of cells from said patient
7. The method of claim 6, wherein said sequencing is conducted
through hybridization or restriction sequencing techniques.
8. The method of claim 1, wherein said determining step comprises
comparing said genotype characteristic to a database of genotype
characteristics associated with resistance to or altered
disposition of said chemotherapeutic agent.
9. A method for selecting a chemotherapeutic agent for treating a
patient, the method comprising: conducting an assay to determine
whether a chemotherapeutic agent effects cellular phenotype of a
sample of cells from a patient; determining whether cells from said
patient comprise a genotypic characteristic associated with
resistance to said chemotherapeutic; and selecting said
chemotherapeutic agent for treating said patient if said
chemotherapeutic agent effects cellular phenotype of said sample of
cells in said conducting step and if said cells in said determining
step do not comprise a genotypic characteristic associated with
resistance to said chemotherapeutic agent.
10. The method of claim 9, wherein said sample of cells in said
conducting step comprise malignant cells.
11. The method of claim 9, wherein said sample of cells in said
conducting step comprise abnormal proliferating cells.
12. The method of claim 9, wherein said cellular phenotype is cell
growth rate or death.
13. The method of claim 9, wherein said genotypic change is a
genetic polymorphism or mutation.
14. The method of claim 9, wherein said determining step comprises
sequencing a portion of the genome of cells from said patient
15. The method of claim 14, wherein said sequencing is accomplished
through hybridization or restriction sequencing techniques.
16. The method of claim 9, wherein said determining step comprises
comparing said genotype characteristics to a database of genotype
characteristics associated with resistance to or altered
disposition of said chemotherapeutic agent.
17. A method for assessing efficacy of a chemotherapeutic agent on
malignant cells in a patient, the method comprising: exposing
malignant cells from a patient to a chemotherapeutic agent;
conducting an assay to determine whether said chemotherapeutic
agent effects cellular phenotype of said malignant cells from said
patient; determining whether a sample of cells from said patient
comprise a genotypic characteristic associated with resistance to
said chemotherapeutic agent; and assessing efficacy of said
chemotherapeutic agent on said malignant cells from said patient
based upon results of said conducting and detecting steps.
18. The method of claim 17, further comprising: selecting said
chemotherapeutic agent for treating said patient if said
chemotherapeutic agent effects cellular phenotype of said malignant
cells in said conducting step and if said cells in said determining
step do not comprise a genotypic characteristic associated with
resistance to said chemotherapeutic agent.
19. The method of claim 17, wherein said malignant cells are
obtained from a tumor specimen from said patient.
20. The method of claim 17, wherein said cells in said determining
step are obtained from a blood sample from said patient.
21. The method of claim 17, wherein said cells in said determining
step are obtained from a buccal smear from said patient.
22. The method of claim 17, wherein said cellular phenotype is cell
growth rate or death.
23. The method of claim 17, wherein said genotypic change is a
genetic polymorphism or mutation.
24. The method of claim 17, wherein said determining step comprises
sequencing a portion of the genome of cells from said patient
25. The method of claim 24, wherein said sequencing is accomplished
through hybridization or restriction sequencing techniques.
26. The method of claim 17, wherein said determining step comprises
comparing said genotype characteristics to a database of genotype
characteristics associated with resistance to or altered
disposition of said chemotherapeutic agent.
Description
RELATED APPLICATIONS
[0001] This application claims priority to U.S. Application Serial
No. 60/417,439, filed Oct. 12, 2002, the disclosure of which is
incorporated by reference herein.
FIELD OF THE INVENTION
[0002] The invention relates to methods for assessing efficacy of
chemotherapeutic agents.
BACKGROUND
[0003] Cancer chemotherapy involves the use of cytotoxic drugs to
destroy unwanted cells in patients. Treatment may consist of using
one or more cytotoxic drugs, depending on the nature of the disease
being treated. However, drug toxicity and drug resistance are
significant barriers effective chemotherapy.
[0004] Toxicity from chemotherapeutic agents produces side effects
ranging from mild trauma to death. Moreover, repeated exposure to
chemotherapeutic drugs is itself often fatal. As chemotherapeutic
drugs are carried in the blood, they are taken up by proliferating
cells, including normal cells. Tissues with high growth rates such
as bone marrow and epithelial tissues, including the
gastrointestinal tract, are normally most susceptible to toxic side
effects. Some drugs have additional toxic effects on other tissues,
such as the urinary tract, myocardium, or pancreas.
Chemotherapeutic agents may cause direct injury to the heart,
either acutely, in the form of myocardial tissue injury or
dysrhythmias, or in a delayed or chronic fashion associated with
congestive heart failure.
[0005] Target cells, such as malignant or diseased cells, may be
intrinsically resistant to chemotherapeutic drugs or they may
acquire resistance as a result of exposure. A target cell may be
genetically predisposed to resistance to particular
chemotherapeutics. Alternatively, the cell may not have receptors
or activating enzymes for the drug or may not be reliant on the
biochemical process with which the drug interferes. Additionally,
individuals may be inherently resistant to a drug due to altered
disposition of the drug in organs other than the tumor. These
mechanisms include, but are not limited to, rapid metabolism to
inactive species, failure to metabolize to an active species of
drug, and rapid clearance or sequestration. Many of these aspects
are encoded genetically by normal polymorphisms in metabolic genes
that act primarily, but not exclusively, in the liver and
gastrointestinal tract and the kidneys.
[0006] Acquired resistance also may develop after cells have been
exposed to a drug or to similar classes of drugs. One example of
acquired drug resistance is the multiple drug resistance phenotype.
Multiple drug resistance is a phenomenon of cross-resistance of
cells to a variety of chemotherapeutic agents which are not
structurally or functionally related. This phenomenon is typically
mediated by p-glycoprotein, a cell membrane pump that is present
normally on the surface of some epithelial cells. The protein
actively removes drug from the cell, making it resistant to drugs
that are substrates for the cell membrane pump.
[0007] A critical issue in cancer chemotherapy is the ability to
select drugs that not only affect cancer cell phenotype in cell
culture assays, but are also not subject to resistance whether in
the tumor or intrinsic to the patient. The present invention
addresses that issue.
SUMMARY OF THE INVENTION
[0008] The invention provides methods for accurately predicting
efficacy of chemotherapeutic agents. Methods of the invention
increase the positive predictive value of chemosensitivity assays
by assessing both the ability of a chemotherapeutic to affect tumor
cells phenotype and the genetic propensity of the patient for
resistance to the chemotherapeutic. Results obtained using methods
of the invention provide insight into the in vivo effectiveness of
a therapeutic, and lead to more effective, individualized,
chemotherapeutic choices.
[0009] According to the invention, a phenotype assay screens a
therapeutic candidate for the ability to affect the phenotype of
tumor cells in culture. A therapeutic candidate that produces the
desired phenotypic effect (e.g., cell death, decreased motility,
changes in cellular adhesion, angiogenesis, or gene expression,
among others) then is screened against genetic properties of cells
of the patient which make resistance to the therapeutic candidate
likely or possible. A therapeutic candidate that has a desired
phenotypic effect on patient tumor cells and that does not appear
to be subject to genetic-based resistance is selected for use. As a
result of combining phenotypic and genetic data, use of the
invention increases the likelihood that a therapeutic candidate,
chosen on the basis of its ability to affect cellular phenotype,
will be effective when administered to patients.
[0010] Accordingly, the invention provides methods for assessing
efficacy of chemotherapeutic agents comprising exposing cells to a
chemotherapeutic agent, conducting an assay to determine whether
the chemotherapeutic agent affects tumor cell phenotype, and
identifying genetic characteristics of cells of the patient (which
may or may not be tumor cells) that indicate a propensity for
resistance to the chemotherapeutic agent.
[0011] In a preferred embodiment, a phenotypic assay for use in the
invention comprises obtaining a tumor explant from a patient,
culturing portions of the explant, growing a monolayer of relevant
cells from the explant, exposing the monolayer to a drug candidate,
and assessing the ability of the drug candidate to alter tumor cell
phenotype. A preferred phenotypic assay is disclosed in U.S. Pat.
No. 5,728,541, and in co-owned, co-pending U.S. application Ser.
No. 10/208,480, both of which are incorporated by reference
herein.
[0012] Genotype analysis according to the invention is accomplished
by any known method. A preferred method comprises comparing the
genotype, or portion thereof, of cells obtained from the patient
with genotypes known to be associated with drug resistance
generally, or specifically with respect to a therapeutic candidate
being evaluated. For example, the existence in patient cells of a
polymorphic variant that is known or suspected to confer resistance
to a therapeutic candidate would screen that candidate out as a
potential therapeutic against those cells. Genetic characteristics
of patient cells are determined by methods known in the art (e.g.,
sequencing, polymorphisms) as set forth below. The impact of a
patient's genotype upon drug resistance may be determined by
reference to genetic databases or libraries that catalog known
mutations or polymorphisms related to resistance.
[0013] The present invention also provides methods for selecting a
chemotherapeutic agent for treating a patient based on results
obtained from the phenotypic and genotypic assays. In a preferred
embodiment, the present invention allows for the assessment of
whether a chemotherapeutic agent will be effective in treating a
cancer when administered to a patient. According to the invention,
chemotherapeutic agents or combinations of chemotherapeutic agents
are selected for treatment where an effect on cellular phenotype is
observed and characteristics of genetic-based resistance are not
observed.
[0014] Methods of the invention are useful in drug or
chemotherapeutic agent screening to provide information indicative
of the in vivo reactivity of the cells, and thus the specific
efficacy as to a particular patient. Methods of the invention are
also useful to screen new drug candidates for therapeutic efficacy
and to provide a basis for categorizing drugs with respect to the
tumor types against which they will work best.
[0015] A phenotypic assay according to the invention is conducted
on cells obtained from a tumor explant from a patient. Genotypic
assays of the invention are performed on genetic data obtained from
patient cells, regardless of their source. Thus, a genotypic assay
can be performed on somatic cells obtained from the patient or on
cells from the same tumor that is evaluated in the phenotypic
assay. Assays of the invention can be performed on an
individualized basis or on a pool of samples obtained from multiple
individual patients. If assays are conducted on pooled samples, the
phenotypic characteristics of the pool of samples are determined
followed by individualized genotypic assays on specific patients.
This allows multiplexing of the phenotypic portion of the
assay.
DETAILED DESCRIPTION OF THE INVENTION
[0016] This invention provides methods for assessing efficacy of
chemotherapeutic agents. Specifically, the invention provides
methods for assessing the efficacy of chemotherapeutic agents based
on phenotypic changes observed in tumor cells obtained from a
patient and genetic characteristics of the patient that indicate
general or specific chemotherapeutic resistance. In one aspect of
the invention, efficacy of a chemotherapeutic agent is assessed
based upon the results of the phenotypic and genotypic assays. In
another aspect of the invention, chemotherapeutic agents are
selected for treating a patient based on the results of the
phenotypic and genotypic assays.
[0017] The present invention is also useful for screening of
therapeutic agents against other diseases, including but not
limited to, hyperproliferative diseases, such as psoriasis. In
addition, the screening of agents that retard cell growth
(anti-cancer, anti-proliferative), including agents that enhance or
subdue intracellular biochemical functions, are evaluated using
methods of the present invention. For example, the effects of
therapeutics on the enzymatic processes, neurotransmitters, and
biochemical pathways are screened using methods of the invention.
Methods of the invention can be practiced on any type of cell
obtained from a patient, including, but not limited to, normal
somatic cells, malignant cells, abnormal proliferating cells, and
other diseased cells. Cells are obtained from any patient sample,
including, but not limited to, tumors, blood samples and buccal
smears. The skilled artisan recognizes that methods of the
invention can be practiced using a variety of different
samples.
[0018] In one step of the invention, a phenotype assay is employed
to assess sensitivity and resistance to chemotherapeutic agents.
The phenotypic assay is performed in vitro using cultured cells.
The phenotype assay allows for identification and separation of
target cells from other cells found in a tissue sample, as well as
direct measurement and monitoring of target cells in response to
chemotherapeutic treatment. Direct measurements and monitoring of
live cells are performed using known methods in the art including,
for example, the measuring of doubling rate, fraction proliferative
assays, monitoring of cytostasis, cell death, cell adhesion, gene
expression, angiogenesis, cell motility, and others. Direct
measurements also include known assays, such as those directed to
measurement and monitoring of apoptosis, senescence, and
necrosis.
[0019] In another step of the invention, a genotype assay is
performed to determine whether cells from a patient comprise a
genetic characteristic associated with resistance to the
chemotherapeutic agents. Genotype assays reveal latent resistance
to chemotherapeutic agents not observed by phenotypic assays.
Genotypic assays may measure characteristics, such as metabolism,
toxic effects, absorption of a therapeutic candidate.
[0020] In one embodiment of the invention, the phenotypic assay is
performed using cell culture monolayers prepared from tumor cells.
In a preferred embodiment, monolayers are cultured from cohesive
multicellular particulates generated from a tumor biopsy. Explants
of tumor tissue sample are prepared non-enzymatically, for initial
tissue culture monolayer preparation. The multicellular tissue
explant is removed from the culture growth medium at a
predetermined time to both allow for the growth of target cells and
prevent substantial growth of non-target cells such as fibroblasts
or stromal cells.
[0021] By way of example, in one embodiment of the invention, a
cell culture monolayer is prepared in accordance with the invention
using the following procedure. A biopsy of non-necrotic,
non-contaminated tissue is obtained from a patient by any suitable
biopsy or surgical procedure known in the art. In a preferred
embodiment, the tissue sample is tumor tissue. The size of the
biopsy sample is not central to the methods provided herein, but a
sample is preferably about 5 to 500 mg, and more preferably about
100 mg. Biopsy sample preparation generally proceeds under sterile
conditions. Cohesive multicellular particulates (explants) are
prepared from the tissue sample by enzymatic digestion or
mechanical fragmentation. Ideally, mechanical fragmentation of the
explant occurs in a medium substantially free of enzymes that are
capable of digesting the explant. For example, the tissue sample
may be minced with sterile scissors to prepare the explants. In a
particularly preferred embodiment, the tissue sample is
systematically minced by using two sterile scalpels in a
scissor-like motion, or mechanically equivalent manual or automated
opposing incisor blades. This cross-cutting motion creates smooth
cut edges on the resulting tissue multicellular particulates. After
the tissue sample has been minced, the particles are plated in
culture flasks (for example, 9 explants per T-25 flask or 20
particulates per T-75 flask). The explants are preferably evenly
distributed across the bottom surface of the flask, followed by
initial inversion for about 10-15 minutes. The flask is then placed
in a non-inverted position in a 37.degree. C. CO.sub.2 incubator
for about 5-10 minutes. In another embodiment in which the tissue
sample comprises brain cells, the flasks are placed in a 35.degree.
C., non-CO.sub.2 incubator. Flasks are checked regularly for growth
and contamination.
[0022] The multicellular explant is removed from the cell culture
at a predetermined time, as described below. Over a period of a few
weeks a monolayer is produced. With respect to the culturing of
tumor cells, it is believed (without any intention of being bound
by the theory) that tumor cells grow out from the multicellular
explant prior to contaminating stromal cells. Therefore, by
initially maintaining the tissue cells within the explant and
removing the explant at a predetermined time, growth of the tumor
cells (as opposed to stromal cells) into a monolayer is
facilitated. The use of the above procedure to form a cell culture
monolayer maximizes the growth of tumor cells from the tissue
sample, and thus optimizes the phenotypic and genotypic assays.
[0023] Once a primary culture and its derived secondary monolayer
tissue culture has been initiated, the growth of the cells is
monitored to oversee growth of the monolayer and ascertain the time
to initiate the phenotypic assay. Prior to the phenotypic assay,
monitoring of the growth of cells may be conducted by visual
monitoring of the flasks on a periodic basis, without killing or
staining the cells and without removing any cells from the culture
flask. Data from periodic counting or measuring is then used to
determine growth rates or cell motility, respectively.
[0024] Phenotypic assays are performed on cultured cells using a
chemotherapeutic drug response assay with clinically relevant dose
concentrations and exposure times. One embodiment of the present
invention contemplates a phenotypic assay that assesses whether
chemotherapeutic agents effect cell growth. Monolayer growth rate
is monitored using, for example, a phase-contrast inverted
microscope. In one embodiment, culture flasks are incubated in a
(5% CO.sub.2) incubator at about 37.degree. C. The flask is placed
under the phase-contrast inverted microscope, and ten fields (areas
on a grid inherent to the flask) are examined using a 10.times.
objective. In general, the ten fields should be non-contiguous, or
significantly removed from one another, so that the ten fields are
a representative sampling of the whole flask. Percentage cell
occupancy for each field examined is noted, and averaging of these
percentages then provides an estimate of overall percent confluency
in the cell culture. When patient samples have been divided between
two more flasks, an average cell count for the total patient sample
should be calculated. The calculated average percent confluency
should be entered into a process log to enable compilation of
data--and plotting of growth curves--over time. Alternatively,
confluency is judged independently for each flask. Monolayer
cultures may be photographed to document cell morphology and
culture growth patterns. The applicable formula is: 1 Percent
confluency = estimate of the area occupied by cells total area in
an observed field
[0025] As an example, therefore, if the estimate of area occupied
by the cells is 30% and the total area of the field is 100%,
percent confluency is {fraction (30/100)}, or 30%.
[0026] Following initial culturing of the multicellular tissue
explant, the tissue explant is removed from the growth medium at a
predetermined time. In one embodiment, the explant is removed from
the growth medium prior to the emergence of a substantial number of
stromal cells from the explant. Alternatively, the explant may be
removed according to the percent confluency of the cell culture. In
one embodiment of the invention, the explant is removed at about 10
to about 50 percent confluency. In a preferred embodiment of the
invention, the explant is removed at about 15 to about 25 percent
confluency. In a particularly preferred embodiment, the explant is
removed at about 20 percent confluency. By removing the explant in
either of the above manners, a cell culture monolayer predominantly
composed of target cells (e.g., tumor cells) is produced. In turn,
a substantial number of non-target cells, such as fibroblasts or
other stromal cells, fail to grow within the culture. Ultimately,
this method of culturing a multicellular tissue explant and
subsequently removing the explant at a predetermined time allows
for increased efficiency in both the preparation of cell cultures
and subsequent phenotypic and genotypic assays for assessing
efficacy of chemotherapeutic agents.
[0027] In another embodiment, a phenotypic assay assesses whether
chemotherapeutic agents effect cell motility. Methods for measuring
cell motility are known by persons skilled in the art. Generally,
these methods monitor and record the changes in cell position over
time. Examples of such methods include, but are not limited to,
video microscopy, optical motility scanning (for example, see U.S.
Pat. No. 6,238,874, the disclosure of which is incorporated by
reference herein) and impedance assays. In a preferred embodiment,
cell motility assays are carried out using monolayer cultures of
malignant cells as described herein.
[0028] Cell culture methods of the invention permit the expansion
of a population of proliferating cells in a mixed population of
abnormal proliferating cells and other (normal) cells. The mixed
population of cells typically is a biopsy or sample from a solid
tumor. A tissue sample from the patient is harvested, cultured and
analyzed for genetic indicia of resistance to chemotherapeutics.
Subcultures of the cells produced by the culture methods described
above may be separately exposed to a plurality of treatments and/or
therapeutic agents for the purpose of objectively identifying the
best treatment for the patient. By way of example, procedures for
culturing the malignant cells and determining a phenotypic to a
chemotherapeutic agent may be performed in the following manner.
First, a specimen is finely minced and tumor fragments are plated
into tissue culture. The cells are then exposed to growth medium,
such as a tumor-type defined media with serum. The cells are
trypsinized, preferably, but not necessarily, when greater than
150,000 cells grown out from tumor fragment. The cells are
preferably plated into a Terasaki plate at 350 cells per well. The
cells are analyzed to verify that a majority of cells are tumor
epithelial cells. Non-adherent cells are removed from the wells.
The cells are treated with 6 concentrations and 2 control lanes of
chemotherapeutic agent or agents for preferably 2 to 4 hours. The
chemotheraputic agents are removed by washing. The cells are
incubated for preferably 3 days. The living cells are counted to
calculate the kill dose that reduces by 40% the number of cells per
well from control wells.
[0029] The culture techniques of the present invention result in a
monolayer of cells that express cellular markers, secreted factors
and tumor antigens in a manner representative of their expression
in vivo. Specific method innovations such as tissue sample
preparation techniques render this method practically, as well as
theoretically, useful.
[0030] According to the present invention, cells from a patient are
analyzed for genetic characteristics (abnormalities) specific to a
patient. Genetic characteristic of a cell or cell population can be
analyzed alone or in combination with other characteristics.
Genetic characteristics of the invention can be, without
limitation, a genetic polymorphism or a mutation, such as an
insertion, inversion, deletion, or substitution. In one embodiment,
nucleic acids are isolated from cells of a patient and analyzed to
identify genotypic characteristics of the cells. The isolated
nucleic acid is DNA or RNA. The nucleic acid, preferably, is
analyzed in a microarray for DNA-encoded polymorphisms in the
coding or control regions of the gene. In another embodiment, the
nucleic acid is analyzed in a microarray for aberrant expression of
one or more genes. In this embodiment, the microarray contains
nucleic acids that are characteristic of known malignancies, as
well as nucleic acids, that are not correlated with known
malignancies so that previously unknown relationships between gene
expression and a proliferative disease or condition may be
identified.
[0031] A preferred method of the invention comprises comparing the
genotype, or portion thereof, of cells from a patient with
genotypes known to be associated with drug resistance generally, or
specifically with respect to a therapeutic candidate being
evaluated. For example, the existence in patient cells of a
polymorphic variant that is known or suspected to confer resistance
to a therapeutic candidate would screen that candidate out as a
potential therapeutic against those cells.
[0032] Methods for isolating and analyzing nucleic acids derived
from the cells are known in the art. The presence of known
proliferation markers, such as the aberrant expression of one or
more genes, the epidermal growth factor receptor (EGFR) cyclin D1,
p16cyclin-kinase inhibitor, retinoblastoma (Rb), transforming
Growth Factor .beta. (TGF.beta.) receptor/smad, MDM2 or p53 genes,
may be determined by, for example, northern blotting or
quantitative polymerase chain reaction (PCR) methods (i.e.,
RT-PCR).
[0033] In one embodiment of the present invention, mRNA
(polyA.sup.+ mRNA) is isolated and labeled cDNA is prepared
therefrom. The labeled cDNA is prepared by synthesizing a first
strand cDNA using an oligo-dT primer, reverse transcriptase and
labeled deoxynucleotides, such as, Cy5-dUTP, commercially available
from Amersham Pharmacia Biotech. Radio-labeled nucleotides also can
be used to prepare cDNA probes. The labeled cDNA is hybridized to
the microarray under sufficiently stringent conditions to ensure
specificity of hybridization of the labeled cDNA to the array
DNA.
[0034] In another embodiment of the invention, the labeled array is
visualized. Visualization of the array may be conducted in a
variety of ways. For instance, when the reading of the microarray
is automated and the labeled DNA is labeled with a fluorescent
nucleotide, the intensity of fluorescence for each discreet DNA of
the microarray can be measured automatically by a robotic device
that includes a light source capable of inducing fluorescence of
the labeled cDNA and a spectrophotometer for reading the intensity
of the fluorescence for each discreet location in the microarray.
The intensity of the fluorescence for each DNA sample in the
microarray typically is directly proportional to the quantity of
the corresponding species of mRNA in the cells from which the MRNA
is isolated. It is possible to label cDNA from two cell types
(i.e., normal and diseased proliferating cells) and hybridize
equivalent amounts of both probe populations to a single microarray
to identify differences in RNA expression for both normal and
diseased proliferating cells. Tools for automating preparation and
analysis of microarray assays, such as robotic microarrayers and
readers, are available commercially from companies such as Gene
Logic and Nanogen and are under development by the NHGRI. The
automation of the microarray analytical process is desirable and,
for all practical purposes necessary, due to the huge number and
small size of discreet sites on the microarray that must be
analyzed.
[0035] In a further embodiment, DNA microarrays are used in
combination with the cell culturing method of the present invention
due to the increased sensitivity of mRNA quantification protocols
when a substantially pure population of cells are used. For their
ease of use and their ability to generate large amounts of data,
microarrays are preferred, when practicable. However, certain other
or additional qualitative assays may be preferred in order to
identify certain markers.
[0036] In another embodiment, the presence of, or absence of,
specific RNA or DNA species are identified by PCR procedures. Known
genetic polymorphisms, translocations, or insertions (i.e.,
retroviral insertions or the insertion of mobile elements, such as
transposons) often can be identified by conducting PCR reactions
with DNA isolated from cells cultured by the methods of the present
invention. Where the sequence anomalies are located in exons, the
genetic polymorphisms may be identified by conducting a PCR
reaction using a cDNA template. Aberrant splicing of RNA precursors
also may be identified by conducting a PCR reaction using a cDNA
template. An expressed translocated sequence may be identified in a
microarray assay, such as the Affymetrix p53 assay.
[0037] In one embodiment, small or single nucleotide substitutions
are identified by the direct sequencing of a given gene by the use
of gene-specific oligonucleotides as sequencing primers. In a
further embodiment, single nucleotide mutations are identified
through the use of allelic discrimination molecular beacon probes,
such as those described in Tyagi, S. and Kromer, F. R. (1996)
Nature Biotech. 14:303-308 and in Tyagi, S. et al, (1998) Nature
Biotech. 16:49-53, the disclosures of each of which are
incorporated by reference herein.
[0038] Genotypic analysis may be based on experimentation or
experience. Sources for such empirical data made be obtained from,
but not limited to clinical records and/or animal tumor transplant
studies. Genetic characteristics found in the patient cells can be
compared to a database containing known tumor genotypes and their
respective resistance to chemotherapeutic agents. In a preferred
embodiment, a database containing genotypes and their respective
drug resistance profile is used to compare genotypic
characteristics of the target cells to resistance to
chemotherapeutic agents in vivo. Computer algorithms are useful for
carrying out pattern matching routines in complex systems, such as
genetic data-mining. A linear regression algorithm, for example,
can be utilized to analyze a database and identify the genotype
most closely matching the genetic characteristics in the patient
cells. In one embodiment, a comparative analysis of genotypes is
performed using a known linear regression algorithm.
[0039] According to the invention, genotypic characteristics of
patient cells are analyzed to establish whether such
characteristics are associated with resistance to chemotherapeutic
agents in vivo. While the above-mentioned genotypic assays are
useful in the analysis of nucleic acids derived from cells produced
by the culture methods embodied in the present invention, numerous
additional methods are known in the general fields of molecular
biology and molecular diagnostics that may be used in place of the
above-referenced methods. Information obtained from genotypic
assays is analyzed to determine efficacy of chemotherapeutic
agents.
[0040] In a further embodiment of the invention, data obtained by
practicing the methods of the invention, including phenotypic,
genotypic and patient outcome information, is stored in databases.
The contents of these databases include, but are not limited to,
observed in vitro phenotypes (disease factors) and genotypes (host
factors). By applying analytical techniques to the stored
information, predictions of chemotherapeutic efficacy can be made.
Methods of the invention allow for the skilled practitioner to
accurately select an effective course of chemotherapy for a
patients, thus reducing the risk of treatment-related trauma and
resistance.
[0041] In one aspect of the invention, a course of chemotherapy is
selected based on results obtained from the phenotypic and
genotypic assays. The present invention allows for the assessment
of the likelihood of whether chemotherapeutic agents will be
effective in treating a malignancy in a patient. A phenotypic assay
in combination with a genotypic assay operates to minimize the risk
of administering to a patient a chemotherapeutic agent or
combinations of chemotherapeutic agents to which the tumor is
resistant. In one aspect of the invention, chemotherapeutic agents
or combinations of chemotherapeutic agents are selected for
treatment where an effect on cellular phenotype is observed and the
genotypic characteristics associated with resistance are not
observed.
[0042] Chemotherapeutic agents that effect cellular phenotype are
potential candidates for use in the patient. Known procedures that
screen for chemotherapeutic agents are time-consuming and
expensive. In one embodiment of the invention, chemotherapeutic
agents that effect cellular phenotype and lack genetic changes
associated with drug resistance are administered to the patient. In
a further embodiment, genotypic characteristics observed in the
genetic assay undergo a comparative analysis to determine if such
characteristics are associated with drug resistance. In another
embodiment, the phenotypic and genotypic assays are performed in
succession, thereby narrowing the scope of the genotypic
comparative analysis, and reducing labor costs and associated
expenses. In one aspect of the invention, when it is determined
that a chemotherapeutic agent effects cellular phenotype and is not
associated with resistance to cells having the genotypic change, a
patient is treated with the chemotherapeutic agent.
[0043] The following examples provide further details of methods
according to the invention. For purposes of exemplification, the
following examples provide details of the use of methods of the
present invention in cancer treatment. Accordingly, while
exemplified in the following manner, the invention is not so
limited and the skilled artisan will appreciate its wide range of
application upon consideration thereof.
EXAMPLE 1
[0044] A patient was diagnosed with breast cancer and
chemotherapeutic treatment was prescribed by the treating
physician. A tumor biopsy of approximately 100 mg of non-necrotic,
non-contaminated tissue was harvested from the patient by surgical
biopsy and transferred to a laboratory in a standard shipping
container. Biopsy sample preparation proceeded as follows. Reagent
grade ethanol was used to wipe down the surface of a Laminar flow
hood. The tumor was then removed, under sterile conditions, from
its shipping container, and cut into quarters with a sterile
scalpel. Using sterile forceps, each undivided tissue quarter was
then placed in 3 ml sterile growth medium (Standard F-10 medium
containing 17% calf serum and a standard amount of Penicillin and
Streptomycin) and minced by using two sterile scalpels in a
scissor-like motion. After each tumor quarter was minced, the
particles were plated in culture flasks using sterile pasteur
pipettes (9 explants per T-25 or 20 particulates per T-75 flask).
Each flask was then labeled with the patient's code and the date of
explantation. The explants were evenly distributed across the
bottom surface of the flask, with initial inverted incubation in a
37.degree. C. incubator for 5-10 minutes, followed by addition of
about 5-10 ml sterile growth medium and further incubation in the
normal, non-inverted position. Flasks were placed in a 35.degree.
C., non-CO.sub.2 incubator. Flasks were checked daily for growth
and contamination as the explants grew out into a cell
monolayer.
[0045] Following initiation of prime cell culture of the tumor
specimen, cells were removed from the monolayers grown in the
flasks for centrifugation into standard size cell pellets. Each
cell pellet was then suspended in 5 ml of the above-described
medium and was mixed in a conical tube with a vortex for 6 to 10
seconds, followed by manual rocking back and forth 10 times. A 36
ml droplet from the center of each tube was then pipetted into one
well of a 96-well microtiter plate together with an equal amount of
trypan blue, plus stirring. The resulting admixture was then
divided between two hemocytometer quadrants for examination using a
standard light microscope. Cells were counted in two out of four
hemocytometer quadrants, under 10.times. magnification--only those
cells which did not take up the trypan blue dye were counted. This
process was repeated for the second counting chamber. An average
cell count per chamber was calculated, and the optimum
concentration of cells in the medium was determined.
[0046] Accommodating the above calculations, additional cell
aliquots from the 4 monolayers were separately suspended in growth
medium via vortex and rocking and were loaded into a Terasaki
dispenser adapted to a 60-well plate. Aliquots of the prepared cell
suspension were delivered into the microtiter plates using Terasaki
dispenser techniques. Cells were plated into 60-well microtiter
plates at a concentration of 100 cells per well.
[0047] Twenty-four hours post-plating, the chemotherapeutic agent
paclitaxel sold under the trademark TAXOL (Bristol-Myers Squibb
Company) was applied to the wells in the microtiter plates. Three
treatment rows in the plates (Rows 2, 3, and 4) were designed to
have escalating paclitaxel doses (1.0, 5.0, and 25 .mu.M). Row 5
served as a control. The paclitaxel exposure time was two hours.
The cells were allowed to incubate for another 72 hours so that
inhibition of cell proliferation can be observed. During this
period, the growth inhibiting effect of paclitaxel was monitored by
observing the percent of confluency of the cells. For each
microtiter well, the percent of confluency of cultured cells was
plotted as a function of time.
[0048] Since paclitaxel affected growth rate of the cultured cells,
cells from the patient were subjected to genotypic analysis. DNA
was isolated from cells of the patient and analyzed for single
nucleotide genetic polymorphisms. Known genetic polymorphisms were
identified in the DNA by conducting PCR reactions and sequencing or
SNP detection by hybridizations of a region of interest in the DNA.
The DNA region of interest from the patient cells was compared to
corresponding regions from known genetic banks and libraries (for
example, GENBANK).
[0049] The phenotypic and genotypic assays were used in combination
to determine that paclitaxel was an efficacious course of treatment
for the patient. As a result, paclitaxel was administered to the
patient.
EXAMPLE 2
[0050] A patient was diagnosed with lung cancer and
chemotherapeutic treatment was prescribed by the treating
physician. A tumor biopsy of approximately 100 mg of non-necrotic,
non-contaminated tissue was harvested from the patient by surgical
biopsy and transferred to a laboratory in a standard shipping
container. The biopsy sample was prepared as described in Example
1. Twenty-four hours post-plating, the chemotherapeutic agent
carboplatin sold under the trademark PARAPLATIN (Bristol-Myers
Squibb Company) was applied to the wells in the microtiter plates.
The first three treatment rows in the plates (Rows 2, 3, and 4)
were designed to have escalating carboplatin doses (50, 200, and
1000 .mu.M). Row 5 serves as a control. The carboplatin exposure
time was two hours. The cells were allowed to incubate for another
72 hours so that inhibition of cell motility can be observed.
[0051] Cell motility was measured by calculating the distance a
cell travels over time. Cells were monitored using a digital
video-camera mounted on a phase-contrast light microscope. To
maintain the growth medium at 35.degree. C., the microscope was
fitted with a heated slide stage. After the cultured cells were
incubated with carboplatin, cell migration was recorded under
appropriate magnification (usually between 40.times. and
200.times.). During this period, the motility inhibiting effect of
carboplatin was documented by plotting the distance cells travel as
a function of time. The distance cells travel was a determined
using digital imaging techniques known in the art.
[0052] Since carboplatin affected cell motility in the tumor cells,
the cells were subjected to genotypic analysis by comparing DNA
from the cultured cells to known genetic banks and libraries. Known
genetic polymorphisms were identified in the cultured cells by
conducting PCR reactions and sequencing a region of interest in DNA
isolated from the cultured cells. The DNA region of interest from
the cultured cells was compared to corresponding regions from known
genetic banks and libraries (for example, GENBANK).
[0053] Genetic characteristcs observed in the genotypic assay were
compared to a database of genetic characteristics that were known
to be associated with resistance to carboplatin. The phenotypic and
genotypic assays were used in combination to determine that
carboplatin was an efficacious course of treatment for the patient.
As a result, carboplatin was administered to the patient.
[0054] While the invention has been shown and described with
reference to specific preferred embodiments, it should be
understood by those skilled in the art that various changes in form
and detail may be made therein without departing from the spirit
and scope of the invention as defined by the following claims.
* * * * *