U.S. patent application number 16/751145 was filed with the patent office on 2020-07-23 for methods of identifying individuals at increased risk of lung cancer.
The applicant listed for this patent is Trustees of Boston University University of Utah Research Foundation. Invention is credited to Andrea Bild, Jerome S. Brody, Adam Gustafson, Avrum Spira.
Application Number | 20200232045 16/751145 |
Document ID | / |
Family ID | 40468411 |
Filed Date | 2020-07-23 |
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United States Patent
Application |
20200232045 |
Kind Code |
A1 |
Brody; Jerome S. ; et
al. |
July 23, 2020 |
METHODS OF IDENTIFYING INDIVIDUALS AT INCREASED RISK OF LUNG
CANCER
Abstract
The invention provides the identification of oncogenic pathways
activated in cytologically normal airway cells of individuals
having or at risk of having lung disease, as well as specific gene
expression patterns (biomarkers) associated with pathway
activation. These biomarkers and pathways may provide prognostic
and/or diagnostic indicators in lung disease, e.g., lung cancer.
Additionally, these pathways and biomarkers may provide therapeutic
targets for the treatment of lung disease, as well as markers for
the assessment of treatment efficacy.
Inventors: |
Brody; Jerome S.; (Boston,
MA) ; Spira; Avrum; (Newton, MA) ; Gustafson;
Adam; (Portland, OR) ; Bild; Andrea; (Salt
Lake City, UT) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Trustees of Boston University
University of Utah Research Foundation |
Boston
Salt Lake City |
MA
UT |
US
US |
|
|
Family ID: |
40468411 |
Appl. No.: |
16/751145 |
Filed: |
January 23, 2020 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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14500475 |
Sep 29, 2014 |
10570454 |
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16751145 |
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12234588 |
Sep 19, 2008 |
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14500475 |
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60994643 |
Sep 19, 2007 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
G01N 2333/91205
20130101; C12Q 1/485 20130101; G01N 2800/50 20130101; A61K 31/5377
20130101; G01N 33/57423 20130101; A61P 35/00 20180101; C12Q 1/6809
20130101; C12Q 2600/106 20130101; A61P 43/00 20180101; C12Q 1/6886
20130101; A61K 31/047 20130101; C12Q 2600/158 20130101; G01N
2800/7028 20130101; A61P 11/00 20180101; C12Q 2600/16 20130101 |
International
Class: |
C12Q 1/6886 20060101
C12Q001/6886; G01N 33/574 20060101 G01N033/574; A61K 31/047
20060101 A61K031/047; A61K 31/5377 20060101 A61K031/5377; C12Q 1/48
20060101 C12Q001/48; C12Q 1/6809 20060101 C12Q001/6809 |
Goverment Interests
GOVERNMENT SUPPORT
[0002] The invention was made with government support under Grant
Nos. NIH/NCI R01CA124640 and NIH/NCI CA106506 awarded by the
National Cancer Institute at the National Institutes of Health. The
government has certain rights in the invention.
Claims
1. A method of identifying an individual at increased risk of lung
disease, comprising determining the activation status of the PI3K
pathway in a cytologically normal airway epithelial cell from said
individual, wherein activation of the PI3K pathway is indicative
that said individual is at increased risk of lung disease as
compared with an individual in whom the PI3K pathway is not
activated.
Description
RELATED APPLICATIONS
[0001] This application is a continuation of U.S. application Ser.
No. 14/500,475, filed on Sep. 29, 2014, which is a continuation of
U.S. application Ser. No. 12/234,588, filed on Sep. 19, 2008, which
claims the benefit of U.S. provisional application Ser. No.
60/994,643, filed Sep. 19, 2007, the entire teachings of which are
incorporated herein by reference.
BACKGROUND OF THE INVENTION
[0003] Cigarette smoke is the dominant cause of lung cancer in the
United States, accounting for an estimated 90% of all cases [1].
However, the damage caused by cigarette smoke is not limited solely
to the lung, but rather constitutes a `field of injury` throughout
the entire respiratory tract [2-6]. An important product of the
field of injury hypothesis is the ability to glean clinically
relevant information from cells collected in regions of the
respiratory tract, such as the bronchial airway, that can be
obtained in a less invasive manner than is typical of collecting
primary lung tissue. Based on this approach, a gene
expression-based biomarker measured in the cytologically normal
bronchial airway epithelium that can distinguish smokers with and
without lung cancer has been developed [7]. This airway gene
expression biomarker achieved 83% accuracy in predicting whether a
smoker had a lung tumor in a prospective test set, and 94% accuracy
when combined synergistically with clinical variables [7, 8].
[0004] Beyond serving as an early diagnostic tool for lung cancer,
gene expression changes in the cytologically normal airway
epithelium have the potential to improve our understanding of the
signaling events deregulated during early stages of lung cancer.
Lung cancer development in humans is a complex process involving
multiple aberrant events that, when accumulated, lead to
deregulation of crucial cell functions, including cell survival and
proliferation. In primary tumors resected from patients with lung
cancer, many signaling pathways have previously been found to be
deregulated, such as p53, RAS and phosphatidylinositol 3-kinase
(PI3K) [9-11]. Additionally, studies in the field of injury and
field cancerization have found that some of the molecular changes
presumed to be early in tumorigenesis are also reflected in
histologically normal cells both neighboring and more distal to the
primary tumor. For example, the same p53 mutation or loss of
heterozygosity at a specific chromosomal region have been
identified throughout the entire respiratory tract [5, 12].
[0005] Insight into the deregulation of oncogenic pathways in
cytologically normal bronchial airway cells from smokers with lung
cancer will help elucidate mechanisms involved in the progression
into malignancy. Furthermore, understanding which pathways are
deregulated could lead to therapeutic and chemoprophylactic
opportunities at the pre-malignant stage of lung cancer.
SUMMARY OF THE INVENTION
[0006] It has been previously demonstrated that gene expression
profiling of cytologically normal bronchial airway epithelium
reflects a field of injury in smokers that can serve as a sensitive
and specific diagnostic biomarker for lung cancer. Using gene
expression signatures defined by in vitro perturbation of specific
oncogenic pathways, as described herein a significant increase of
phosphatidylinositol 3-kinase (PI3K) pathway activity was
identified in the cytologically normal airway of smokers with lung
cancer (n=129), as well as in lung tumor tissue (n=107). To
evaluate whether increased activity of PI3K occurs prior to the
development of lung cancer, cytologically normal airway epithelia
in high-risk smokers with moderate-severe dysplastic lesions in
their airway (n=14) were profiled, and higher levels of PI3K
pathway gene expression were found as compared to the airway
epithelium from healthy smokers without dysplasia (n=11). Further,
PI3K activity was decreased in the airway of high-risk smokers who
had significant regression of dysplasia following treatment with
the chemoprophylactic agent myo-inositol (n=10). In vitro dilution
experiments confirmed that myo-inositol inhibits the PI3K pathway,
which not only proposes a mechanism of action for myo-inositol, but
also reflects a potential therapeutic relationship between PI3K
pathway activity and regression of airway dysplasia. Together,
these findings suggest that deregulation of the PI3K pathway is an
early, measurable and reversible step in the development of lung
cancer, and that airway gene expression profiling in smokers may
enable personalized approaches to chemoprophylaxis and therapy.
[0007] In one embodiment the invention provides biomarkers for
oncogenic pathways activated in cytologically normal airway
epithelial cells of individuals with lung disease. These biomarkers
and pathways may provide prognostic and/or diagnostic indicators of
lung disease, e.g., lung cancer. Additionally, these pathways and
biomarkers may provide therapeutic targets for the treatment of
lung disease, as well as markers for the assessment of treatment
efficacy.
[0008] In one aspect the invention relates to the use of gene
expression profiling methods to identify gene expression signatures
of oncogenic pathways activated in lung disease, e.g., lung cancer.
These gene expression signatures may provide prognostic or
diagnostic indicators for lung disease, e.g., lung cancer.
Moreover, oncogenic pathways which are activated in lung disease
may be targets for therapeutic intervention. In one embodiment the
oncogenic pathway can be, for example, one or more of the PI3K and
Np63 pathways.
[0009] The invention also relates to a method of identifying an
individual at increased risk of lung disease, comprising
determining the activation status of the Np63 and/or PI3K pathway
in a cytologically normal airway epithelial cell from said
individual, wherein activation of the Np63 and/or PI3K pathway is
indicative that said individual is at increased risk of lung
disease as compared with an individual in whom the Np63 and/or PI3K
pathway is not activated. In particular embodiments the individual
is a smoker or a non-smoker. In other embodiments the lung disease
is lung cancer.
[0010] In one embodiment the activation status of the PI3K pathway
is determined using gene expression data for one or more biomarkers
of the PI3K pathway. For example, in some embodiments at least one
of said one or more biomarkers is a gene which is increased upon
PI3K activation, and in other embodiments at least one of said one
or more biomarkers is a gene which is decreased upon PI3K
activation. Combinations of biomarkers which are increased and
decreased upon PI3K activation may also be used. In particular
embodiments at least one of said one or more biomarkers is a gene
which is upstream of PI3K activation, while in other embodiments at
least one of said one or more biomarkers is a gene which is
downstream of PI3K activation.
[0011] In particular embodiments, expression data for said one or
more biomarkers of the PI3K pathway is obtained using an
oligonucleotide microarray. In other embodiments the activation
status of the PI3K pathway is determined using one or more gene
expression products of one or more biomarkers of the PI3K pathway.
Said gene expression products may be nucleotide or amino acid
products and can be detected using methods known in the art.
[0012] In some embodiments of the invention the activation status
of the PI3K pathway is determined by assessing the activation of
IGF1R, wherein activation of IGF1R is indicative of activation of
the PI3K pathway. In other embodiments of the invention the
activation status of the PI3K pathway is determined by assessing
the activation of PKC, wherein activation of PKC is indicative of
activation of the PI3K pathway.
[0013] The invention also relates to a method of identifying an
individual at increased risk of lung disease, comprising
determining the activation status of PKC in a cytologically normal
airway epithelial cell from said individual, wherein activation of
PKC is indicative that said individual is at increased risk of lung
disease as compared with an individual in whom PKC is not
activated.
[0014] The invention further relates to a method of identifying an
individual at increased risk of lung disease, comprising
determining the activation status of IGF1R in a cytologically
normal airway epithelial cell from said individual, wherein
activation of IGF1R is indicative that said individual is at
increased risk of lung disease as compared with an individual in
whom IGF1R is not activated.
[0015] In other embodiments the invention provides an
oligonucleotide array having immobilized thereon one or more probes
for one or more biomarkers of the PI3K pathway, and wherein said
array does not have immobilized thereon probes for other
biomarkers. In preferred embodiments said one or more biomarkers of
the PI3K pathway are selected from the group consisting of IGF1R,
PKC, the biomarkers disclosed in [29], and combinations
thereof.
[0016] The invention also relates to a method of reducing the risk
of lung disease in an individual comprising administering to an
individual at risk of lung disease one or more agents (e.g., one or
more agents, regimens or treatments or combinations thereof) which
inhibit the PI3K pathway. In particular embodiments the PI3K
pathway is activated in said individual prior to administration of
said one or more agents. In one embodiment the lung disease is lung
cancer. In another embodiment said one or more agents are
administered to said individual prophylactically before the
development of lung disease.
[0017] In another embodiment the invention relates to a method of
differentially classifying a cytologically normal test airway
epithelial cell, comprising identifying a gene expression signature
associated with activation of a biological pathway of interest in a
normal airway epithelial cell; assessing gene expression in
differentially classified airway epithelial cells to identify one
or more correlations between classification of an airway epithelial
cell and activation of a biological pathway of interest; and
assessing gene expression in a cytologically normal test airway
epithelial cell, wherein the gene expression profile of the
cytologically normal airway epithelial cell to be classified
indicates whether the biological pathway of interest is activated
and thus differentially classifies the cell.
[0018] In particular embodiments the biological pathway of interest
is an oncogenic pathway. In some embodiments the differential
classification is increased risk of disease versus decreased risk
of disease, while in other embodiments the differential
classification is response to treatment versus non-response to
treatment.
[0019] In one embodiment, the invention provides a method for
identifying the activation of an oncogenic pathway in a mammal
having or at risk of having lung disease, e.g., lung cancer. The
method may include: (a) providing a biological sample, e.g., a
biological sample from an airway passage of the mammal, wherein the
biological sample comprises a gene expression product (e.g., mRNA
or protein) from at least one gene that is indicative of activation
of said pathway, and (b) detecting the expression of said gene. For
example, the pathway can be one or more of the following: Ras, Myc,
E2F3, beta-catenin, Src, Np63, PI3K and combinations thereof. The
mammal can be, for example, a human. Biological samples may be
provided, for example, from bronchial, nasal or buccal epithelium,
or from biopsied tissue samples. In one embodiment, detection of
gene expression is accomplished using an oligonucleotide array
having immobilized thereon one or more nucleotide sequences or
fragments thereof which are probes for the relevant gene(s).
Identification of activation of an oncogenic pathway may indicate
that the mammal is a candidate for treatment to inhibit activation
of said pathway or for additional or more frequent screening to
identify development of disease, e.g., cancer.
[0020] In another embodiment, the invention provides a method of
screening candidate therapeutic agents which may be useful in the
treatment of lung disease, e.g., lung cancer. For example,
candidate agents may be screened for their ability to modulate
(e.g., inhibit) the activation of an oncogenic pathway identified
by methods described herein as associated with lung disease. The
agent's ability to modulate activation of the pathway may be
assessed, for example, by its ability to alter a gene expression
signature of an oncogenic pathway from a signature which is
associated with disease to a signature which is not associated with
disease, e.g., is normal. Alternatively the candidate therapeutic
agent may be assessed for its ability to modulate a specific
functional effect or readout of the pathway. Agents identified as
having the ability to inhibit an activated oncogenic pathway
associated with lung disease may be suitable for treatment of lung
disease in a mammal.
[0021] In addition, the efficacy of a treatment regimen can be
evaluated by assessing the gene expression signature of a mammal at
various time points over the course of treatment. A shift in the
gene expression signature of an oncogenic pathway from one
associated with disease to one not associated with disease, e.g.,
to a normal signature, is indicative of efficacious treatment.
Similarly absence of a shift in the gene expression signature
toward a normal signature is indicative that treatment is not
efficacious and that perhaps alternative treatment regimens are
indicated.
BRIEF DESCRIPTION OF THE DRAWINGS
[0022] FIGS. 1A-1C show that PI3K and .DELTA.Np63 are
differentially activated in smokers with lung cancer.
[0023] Using binary regression models trained on in vitro gene
expression signatures, pathway activation probabilities were
calculated in samples obtained from the cytologically normal
airway. Pathway levels are summarized using box plots, where the
bar represents the median value, the box denotes the range of the
data points from the 25th to 75th percentile, and the whiskers
specify the range of the remaining 1st and 4th quartile. As shown
in FIG. 1A, when grouping the activation levels by lung cancer
status (blue for no lung cancer, red for lung cancer), two pathways
were found to be statistically different after random permutation
tests: PI3K (p<0.001), and .DELTA.Np63 (p<0.001). To account
for variables that could possibly confound the observed differences
in pathway activation seen in the airway of smokers with lung
cancer, pathway activation probabilities were also calculated for
healthy never (green), former (brown) and current smokers (gray)
(FIG. 1B), as well as current smokers with (orange) or without
(gray) chronic obstructive pulmonary disease (COPD) (FIG. 1C).
Neither of the potential confounding variables showed a
statistically significant difference in pathway activation.
[0024] FIG. 2 shows oncogenic pathway activity in lung tumor and
adjacent normal tissue. Oncogenic pathway activity was calculated
for a dataset of lung adenocarcinoma and adjacent normal tissue
[32]. An increase in PI3K (p<0.001) and .DELTA.Np63 (p=0.002)
was observed when comparing adjacent normal and its paired tumor
sample. Increases were also seen in Myc and E2F3, and there was a
decrease in Src. Error whiskers are reported as SEM.
[0025] FIGS. 3A-3B show the biochemical validation of PI3K activity
in prospectively collected airway samples. Airway brushings were
collected prospectively from patients under suspicion of having
lung cancer in Boston and Utah. As shown in FIG. 3A, kinase assays
were used to measure in vivo levels of PI3K pathway activity.
Patients with lung cancer generally had higher levels of PI3K
activity than those without lung cancer. A subset of the Boston
cohort had extra sample run on microarray so that computational
predicted PI3K activity could be correlated to in vivo activity.
The probability of pathway activity is shown below the patients
that had extra samples. Pearson correlation of the computationally
predicted PI3K activity and the biochemically measured activity was
0.48. As shown in FIG. 3B, Western blots querying proteins both
upstream and downstream of PI3K were quantified and then correlated
with PI3K kinase levels measured in FIG. 3A. Correlations are
presented in a heatmap manner, where blue represents negative
correlation, and red represents positive correlation. Correlation
analysis is also broken down into all samples, only samples with
lung cancer, and only control samples. Both p-IGF1R and p-PKC are
positively correlated with PI3K activity in patients with lung
cancer, suggesting possible sub-pathways driving the increased PI3K
pathway activity in the airway of smokers with lung cancer.
[0026] FIG. 4 demonstrates that smokers with dysplasia have an
increased activation of the PI3K pathway. Genes that increase when
the PI3K pathway is activated, as defined by in vitro perturbation,
are displayed in a heatmap. Blue represents low expression of a
gene, while red represents higher expression of a gene. When
comparing the expression levels of these genes in the cytologically
normal bronchial airway of smokers with dysplasia against healthy
smokers, an increased activation of the PI3K pathway in smokers
with dysplasia was observed. GSEA was used to quantify the
enrichment of this gene set (p<0.001, FDR Q<0.001). Genes
were ranked for GSEA using a linear model that takes into account
dysplasia status, pack-years as well as a random variable
accounting for batch effects.
[0027] FIG. 5A-5C show that Myo-inositol inhibits the PI3K pathway
in vitro. Insulin was used to activate the PI3K pathway in three
different cell lines: (FIG. 5A) BEAS-2B (bronchial airway cell
line), (FIG. 5B) BT549 (breast cancer cell line), and (FIG. 5C)
HEK293 (human embryonic kidney cell line). The cell lines were then
treated with varying doses of myo-inositol and LY-294002. PIP3
levels were measured to quantify the activation levels of the PI3K
pathway (y-axis). In each cell line tested, there was a drop in
PIP3 levels following treatment with either myo-inositol or
LY-294002 (a known PI3K inhibitor), suggesting that myo-inositol
inhibits the PI3K pathway in vitro. Replication of these
experiments produced similar results.
DETAILED DESCRIPTION OF THE INVENTION
[0028] Based on the concept that genomic changes in the epithelial
cells that line the entire respiratory tract reflect host response
to and damage from cigarette smoke, a better understanding of the
early events leading to tumorigenesis may be gained by identifying
which pathways are deregulated in the airway of smokers with or at
risk for having lung cancer. One approach to assess pathway
activity uses gene expression data to link in vitro activation of
an isolated signaling pathway to predict status of that pathway in
patient samples. This approach has been successful at predicting
pathway status in cell lines as well as tumors where the initiation
event is known [13]. A strength of in vitro defined pathway
signatures is that they are capable of identifying pathway activity
at the gene expression level, allowing the measurement of multiple
pathways using a single microarray experiment. Further, gene
expression based predictions of pathway activity have been found to
correlate significantly to drugs that target the specific pathway
[13-20]. Numerous studies have also found correlation of predicted
pathway status and therapeutic responsiveness in clinical trials
with targeted therapies [22-25].
[0029] Work described herein utilized expression signatures
developed through in vitro perturbation. Metagene models were
trained to compare oncogenic pathway activity in the cytologically
normal airway epithelium of smokers with and without lung cancer.
As described herein, using the gene-expression based pathway
approach described above, the data show that the PI3K pathway has
an increased level of activity in cytologically normal bronchial
airway cells of smokers with lung cancer, as well as higher levels
in the lung tumor tissue itself. Biochemical assays measuring in
vivo PI3K activity in a prospectively collected cohort of airway
samples from patients with and without lung cancer validated the
computational predictions. An exploration of the expression
profiles from the cytologically normal airway of high-risk smokers
with dysplastic lesions in their airway again revealed an increased
activity of PI3K. As dysplasia is considered a pre-neoplastic
event, this is suggestive that PI3K levels increased before the
development of lung cancer. Providing possible therapeutic
relevance to this result, high-risk subjects responsive to the
chemoprophylactic agent myo-inositol show a significant reduction
in PI3K activity and regression of dysplastic lesions. The
relationship between myo-inositol and PI3K was further elucidated
by showing that myo-inositol inhibits PI3K in vitro.
[0030] Together, these results demonstrate that the PI3K pathway is
activated in the cytologically normal airway epithelium prior to
the development of lung cancer, and the levels of this pathway
associate with response to chemoprophylaxis with myo-inositol. More
broadly, these findings suggest that airway gene-expression
reflects perturbation of specific oncogenic pathways within a
smoker, potentially allowing for personalized approaches to
chemoprophylaxis and therapy.
[0031] PI3K and .DELTA.Np63 pathways have an increased activation
in the normal airway of smokers with lung cancer (FIG. 1A). This is
an intriguing finding, because a priori cytologically normal cells
are not expected to show signs of oncogenic pathway deregulation.
Additionally, it is important to note that non-lung cancer controls
used as described herein have an extensive range of alternative
pathologies that could also impact the PI3K pathway, and are not
just healthy volunteers. However, the increased activity is not
correlated to smoking status or COPD (FIG. 1B, 10).
[0032] Pre-neoplastic increases in the PI3K pathway were also seen
in the cytologically normal airway of high-risk smokers with
dysplastic airway lesions when compared to healthy smokers. This
supports the hypothesis that PI3K activity is induced prior to the
development of lung neoplasms. In addition, there are higher levels
of PI3K activity in lung tumors as compared to adjacent normal
tissue (FIG. 2), suggesting a further increase in PI3K activity as
cells transform.
[0033] Previous studies using mouse models of lung adenocarcinoma
have shown that PI3K is required for malignant progression in lung
cancer, and that inhibition of this pathway blocks tumorigenesis
[40]. Increased activity of the PI3K pathway has also previously
been observed in many different cancers, including lung cancer
[41]. Some of the common causes of deregulation that confer
constitutive activation in tumors include a mutation in the
tyrosine kinase domain of EGFR; a mutation, deletion or suppression
of the tumor suppressor PTEN; increased PI3K gene copy number [42]
or a mutation in p110.alpha., the catalytic subunit of PI3K
[41].
[0034] In the studies described herein, PI3K activity in the normal
airway epithelium of lung cancer patients is positively correlated
with activation of IGF1R (upstream) and PKC (downstream), and is
not positively correlated with HER2 (upstream) and AKT (downstream)
(FIG. 3). These results suggest a specific signaling cascade
leading to PI3K activation and subsequent downstream effects in
these cells. Increased levels of IGF signaling have been associated
with lung cancer in some studies. Further, current inhibitors of
the IGF pathway have been found to have significant responses in
lung cancer patients. PKC is a kinase downstream of PI3K, and has
previously been found to have increased levels in dysplastic
lesions and lung cancer [33, 34]. Together, results described
herein implicate the IGFR1/PI3K/PKC pathway as central to lung
cancer development, even at a pre-malignant state.
[0035] Of clinical importance is whether a reduction in PI3K levels
prior to the development of lung cancer would offer any therapeutic
potential. Current PI3K pathway inhibitors on the market, such as
sirolimus, have harmful side effects that would prohibit their use
as a long-term prevention option. To help address this crucial
question, a study was conducted on a high-risk cohort that had
undergone treatment with a lung cancer chemoprophylactic agent
called myo-inositol, which has previously been found to reduce
dysplastic lesions in the airway following oral treatment for 2-3
months [35]. In contrast to sirolimus, myo-inositol has the
potential to be taken orally for long periods of time because it
causes very minor side effects. In patients from this cohort that
had responded to myo-inositol as evidenced by a regression of
dysplasia in their airway, gene expression patterns were observed
that reflect a reduction in PI3K activity. Given the relatively
limited samples size for this study (n=10), in vitro studies were
conducted, and it was found that myo-inositol directly inhibits
PI3K activity, suggesting a possible mechanism of action for this
compound (FIG. 5). If the chemoprophylactic properties of
myo-inositol are confirmed in larger clinical trials, use of this
compound in high-risk smokers with perturbed PI3K activity in the
airway could decrease lung cancer occurrence. Airway gene
expression profiling on these subjects post-treatment may also help
identify a subset of patients that would benefit from long-term
therapy. More broadly, these results suggest that a smoker's
pattern of airway gene-expression reflects perturbation of specific
oncogenic pathways, potentially allowing for personalized
chemoprophylaxis and therapy.
[0036] In principal, there are multiple hypotheses that could
explain the increased activity of oncogenic pathways in
cytologically normal airway epithelium. Importantly, these concepts
are not mutually exclusive and likely work in a synergistic manner
to promote disease. First, deregulation could be caused by a
genetic predisposition to lung cancer, such as oncogenic germ-line
mutations. Second, following the field of injury hypothesis,
cigarette smoke exposure damages the entire respiratory tract, and
the damage, such as somatic mutations, could be the source of
oncogenic activity in the airway. The susceptibility to damage will
partly depend on host response to cigarette smoke, which will be
influenced by oncogenic germ-line mutations. Finally, somatic
mutations conferring growth advantages could cause increased
oncogenic activity in the airway due to clonal expansion.
[0037] Cumulatively, these studies successfully use a computational
approach to identify the signaling pathways driving lung cancer
oncogenesis, and to identify rational targeted therapeutic
approaches which may be preventative of cancer development in
high-risk populations. Further, our biochemical measurements
correlate with computational analysis in patient samples, and
highlight the specificity and sensitivity of this approach.
Verification of pathway activation is also seen in in vitro and in
vivo studies linking myo-inositol treatment to inhibition of the
PI3K pathway. This suggests that the deregulation of the PI3K
pathway is an early, measurable and reversible step in the
development of lung cancer and may serve to guide chemopreventative
approaches in high-risk smokers.
[0038] Accordingly the invention provides a general approach to
identifying pathway status (e.g., oncogenic pathway status) in
cytologically normal cells of the airway which may be useful as an
early predictor of lung disease and/or which may provide targets
for therapeutic intervention (e.g., early intervention). According
to this approach, oncogenic or other pathways of interest are
activated in a cell (e.g., human epithelial cell, human primary
epithelial cell culture) in vitro to identify gene expression
signatures or patterns which are associated with pathway
activation. For example, cells can be perturbed using an adenovirus
expressing an activating or necessary component of the pathway of
interest (e.g., an adenovirus expressing p110 or other suitable
agent). Differentially classified samples (e.g., a sample from a
lung cancer patient v. sample from an individual without lung
cancer, a sample from a treatment responsive patient v. a sample
from a treatment non-responsive patient, etc.) can then be assessed
to identify class associations with a gene expression profile
indicative of pathway activation. That is, pathway status (e.g.,
activation) is correlated with phenotype (e.g., disease state,
treatment response, etc.). Thereafter histologically normal airway
cells can be tested to identify a gene expression pattern
associated with activation of a particular pathway, and based on
the correlation between pathway status and phenotype, the phenotype
(e.g., disease state such as cancerous, non-cancerous) of the
individual from whom the cell sample is obtained can be predicted.
In this manner associations between disease state and pathway
status (as indicated by gene expression) can be identified, and
these associations can be leveraged in therapeutic and prognostic
applications. Similarly, the impact of candidate agents and
treatment regimens on activation of one or more pathways can be
assessed by monitoring gene expression associated with said
pathway(s) to identify agents and/or regimens having a desired
effect.
[0039] The invention also relates to a method of identifying an
individual at increased risk of lung disease, comprising
determining the activation status of an oncogenic pathway, e.g.,
the Np63 and/or PI3K pathway, in a cytologically normal airway
epithelial cell from said individual. Activation of, e.g., the Np63
and/or PI3K pathway is indicative that said individual is at
increased risk of lung disease as compared with an individual in
whom the Np63 and/or PI3K pathway is not activated. In particular
embodiments the individual is a smoker or a non-smoker. In other
embodiments the lung disease is lung cancer. I some embodiments the
activation status of multiple pathways (e.g., oncogenic pathways)
is assessed simultaneously.
[0040] In one embodiment the activation status of the PI3K pathway
is determined using gene expression data for one or more (i.e., 1,
2, 3, 4, 5 or more than 5) biomarkers of the PI3K pathway. For
example, in some embodiments at least one of said one or more
biomarkers is a gene which is increased upon PI3K activation, and
in other embodiments at least one of said one or more biomarkers is
a gene which is decreased upon PI3K activation. Combinations of
biomarkers which are increased and decreased upon PI3K activation
may also be used. In particular embodiments at least one of said
one or more biomarkers is a gene which is upstream of PI3K
activation, while in other embodiments at least one of said one or
more biomarkers is a gene which is downstream of PI3K
activation.
[0041] In one embodiment of the present invention, the isolated
nucleic acid is obtained from a cytologically normal airway
epithelial cell and used to evaluate expression of a gene or
multiple genes using any method known in the art for measuring gene
expression, including analysis of mRNA transcripts as well as
analysis of DNA methylation.
[0042] Methods for assessing mRNA levels are well known to those
skilled in the art. In one preferred embodiment, gene expression
can be determined by detection of RNA transcripts, for example by
Northern blotting, for example, wherein a preparation of RNA is run
on a denaturing agarose gel, and transferred to a suitable support,
such as activated cellulose, nitrocellulose or glass or nylon
membranes. Labeled (e.g. radiolabeled) cDNA or RNA is then
hybridized to the preparation, washed and analyzed using methods
well known in the art, such as autoradiography.
[0043] Detection of RNA transcripts can further be accomplished
using known amplification methods. For example, it is within the
scope of the present invention to reverse transcribe mRNA into cDNA
followed by polymerase chain reaction (RT-PCR); or, to use a single
enzyme for both steps as described in U.S. Pat. No. 5,322,770, or
reverse transcribe mRNA into cDNA followed by symmetric gap ligase
chain reaction (RT-AGLCR) as described by R. L. Marshall, et al.,
PCR Methods and Applications 4: 80-84 (1994).
[0044] Other known amplification methods which can be utilized
herein include but are not limited to the so-called "NASBA" or
"3SR" technique described in PNAS USA 87: 1874-1878 (1990) and also
described in Nature 350 (No. 6313): 91-92 (1991); Q-beta
amplification as described in published European Patent Application
(EPA) No. 4544610; strand displacement amplification (as described
in G. T. Walker et al., Clin. Chem. 42: 9-13 (1996) and European
Patent Application No. 684315; and target mediated amplification,
as described by PCT Publication WO 9322461.
[0045] In situ hybridization visualization may also be employed,
wherein a radioactively labeled antisense RNA probe is hybridized
with a thin section of a biopsy sample, washed, cleaved with RNase
and exposed to a sensitive emulsion for autoradiography. The
samples may be stained with haematoxylin to demonstrate the
histological composition of the sample, and dark field imaging with
a suitable light filter shows the developed emulsion.
Non-radioactive labels such as digoxigenin may also be used.
[0046] Alternatively, RNA expression, including MRNA expression,
can be detected on a DNA array, chip or a microarray.
Oligonucleotides corresponding to a gene(s) of interest are
immobilized on a chip which is then hybridized with labeled nucleic
acids of a test sample obtained from a patient. Positive
hybridization signal is obtained with the sample containing
transcripts of the gene of interest. Methods of preparing DNA
arrays and their use are well known in the art. (See, for example
U.S. Pat. Nos. 6,618,6796; 6,379,897; 6,664,377; 6,451,536;
548,257; U.S. 20030157485 and Schena et al. 1995 Science
20:467-470; Gerhold et al. 1999 Trends in Biochem. Sci. 24,
168-173; and Lennon et al. 2000 Drug discovery Today 5: 59-65,
which are herein incorporated by reference in their entirety).
Serial Analysis of Gene Expression (SAGE) can also be performed
(See for example U.S. Patent Application 20030215858).
[0047] The methods of the present invention can employ solid
substrates, including arrays in some preferred embodiments. Methods
and techniques applicable to polymer array synthesis have been
described in U.S. Ser. No. 09/536,841, WO 00/58516, U.S. Pat. Nos.
5,143,854, 5,242,974, 5,252,743, 5,324,633, 5,384,261, 5,405,783,
5,424,186, 5,451,683, 5,482,867, 5,491,074, 5,527,681, 5,550,215,
5,571,639, 5,578,832, 5,593,839, 5,599,695, 5,624,711, 5,631,734,
5,795,716, 5,831,070, 5,837,832, 5,856,101, 5,858,659, 5,936,324,
5,968,740, 5,974,164, 5,981,185, 5,981,956, 6,025,601, 6,033,860,
6,040,193, 6,090,555, 6,136,269, 6,269,846 and 6,428,752, in PCT
Applications Nos. PCT/US99/00730 (International Publication Number
WO 99/36760) and PCT/US01/04285, which are all incorporated herein
by reference in their entirety for all purposes.
[0048] Patents that describe synthesis techniques in specific
embodiments include U.S. Pat. Nos. 5,412,087, 6,147,205, 6,262,216,
6,310,189, 5,889,165, and 5,959,098.
[0049] Nucleic acid arrays that are useful in the present invention
include, but are not limited to those that are commercially
available from Affymetrix (Santa Clara, Calif.) under the brand
name GeneChip7. Example arrays are shown on the website at
affymetrix.com.
[0050] The present invention also contemplates many uses for
polymers attached to solid substrates. These uses include gene
expression monitoring, profiling, library screening, genotyping and
diagnostics. Examples of gene expression monitoring, and profiling
methods are shown in U.S. Pat. Nos. 5,800,992, 6,013,449,
6,020,135, 6,033,860, 6,040,138, 6,177,248 and 6,309,822. Examples
of genotyping and uses therefore are shown in U.S. Ser. No.
60/319,253, 10/013,598, and U.S. Pat. Nos. 5,856,092, 6,300,063,
5,858,659, 6,284,460, 6,361,947, 6,368,799 and 6,333,179. Other
examples of uses are embodied in U.S. Pat. Nos. 5,871,928,
5,902,723, 6,045,996, 5,541,061, and 6,197,506.
[0051] To monitor mRNA levels, for example, mRNA is extracted from
the biological sample to be tested, reverse transcribed, and
fluorescent-labeled cDNA probes are generated. The microarrays
capable of hybridizing to the gene of interest are then probed with
the labeled cDNA probes, the slides scanned and fluorescence
intensity measured. This intensity correlates with the
hybridization intensity and expression levels.
[0052] In one preferred embodiment, gene expression is measured
using quantitative real time PCR. Quantitative real-time PCR refers
to a polymerase chain reaction which is monitored, usually by
fluorescence, over time during the amplification process, to
measure a parameter related to the extent of amplification of a
particular sequence. The amount of fluorescence released during the
amplification cycle is proportional to the amount of product
amplified in each PCR cycle.
[0053] The present invention also contemplates sample preparation
methods in certain preferred embodiments. Prior to or concurrent
with expression analysis, the nucleic acid sample may be amplified
by a variety of mechanisms, some of which may employ PCR. See,
e.g., PCR Technology: Principles and Applications for DNA
Amplification (Ed. H. A. Erlich, Freeman Press, NY, N.Y., 1992);
PCR Protocols: A Guide to Methods and Applications (Eds. Innis, et
al., Academic Press, San Diego, Calif., 1990); Mattila et al.,
Nucleic Acids Res. 19, 4967 (1991); Eckert et al., PCR Methods and
Applications 1, 17 (1991); PCR (Eds. McPherson et al., IRL Press,
Oxford); and U.S. Pat. Nos. 4,683,202, 4,683,195, 4,800,159
4,965,188, and 5,333,675, and each of which is incorporated herein
by reference in their entireties for all purposes. The sample may
be amplified on the array. See, for example, U.S. Pat. No.
6,300,070 and U.S. patent application Ser. No. 09/513,300, which
are incorporated herein by reference.
[0054] Other suitable amplification methods include the ligase
chain reaction (LCR) (e.g., Wu and Wallace, Genomics 4, 560 (1989),
Landegren et al., Science 241, 1077 (1988) and Barringer et al.
Gene 89:117 (1990)), transcription amplification (Kwoh et al.,
Proc. Natl. Acad. Sci. USA 86, 1173 (1989) and WO88/10315),
self-sustained sequence replication (Guatelli et al., Proc. Nat.
Acad. Sci. USA, 87, 1874 (1990) and WO90/06995), selective
amplification of target polyhucleotide sequences (U.S. Pat. No.
6,410,276), consensus sequence primed polymerase chain reaction
(CP-PCR) (U.S. Pat. No. 4,437,975), arbitrarily primed polymerase
chain reaction (AP-PCR) (U.S. Pat. Nos. 5,413,909, 5,861,245) and
nucleic acid based sequence amplification (NABSA). (See, U.S. Pat.
Nos. 5,409,818, 5,554,517, and 6,063,603, each of which is
incorporated herein by reference). Other amplification methods that
may be used are described in, U.S. Pat. Nos. 5,242,794, 5,494,810,
4,988,617 and in U.S. Ser. No. 09/854,317, each of which is
incorporated herein by reference.
[0055] Additional methods of sample preparation and techniques for
reducing the complexity of a nucleic sample are described, for
example, in Dong et al., Genome Research 11, 1418 (2001), in U.S.
Pat. Nos. 6,361,947, 6,391,592 and U.S. patent application Ser.
Nos. 09/916,135, 09/920,491, 09/910,292, and 10/013,598.
[0056] Methods for conducting polynucleotide hybridization assays
have been well developed in the art. Hybridization assay procedures
and conditions will vary depending on the application and are
selected in accordance with the general binding methods known
including those referred to in: Maniatis et al. Molecular Cloning:
A Laboratory Manual (2.sup.nd Ed. Cold Spring Harbor, N.Y., 1989);
Berger and Kimmel Methods in Enzymology, Vol. 152, Guide to
Molecular Cloning Techniques (Academic Press, Inc., San Diego,
Calif., 1987); Young and Davism, P.N.A.S, 80: 1194 (1983). Methods
and apparatus for carrying out repeated and controlled
hybridization reactions have been described, for example, in U.S.
Pat. Nos. 5,871,928, 5,874,219, 6,045,996 and 6,386,749, 6,391,623
each of which are incorporated herein by reference.
[0057] The present invention also contemplates signal detection of
hybridization between ligands in certain preferred embodiments.
See, for example, U.S. Pat. Nos. 5,143,854, 5,578,832; 5,631,734;
5,834,758; 5,936,324; 5,981,956; 6,025,601; 6,141,096; 6,185,030;
6,201,639; 6,218,803; and 6,225,625, in provisional U.S. Patent
application 60/364,731 and in PCT Application PCT/US99/06097
published as WO99/47964), each of which also is hereby incorporated
by reference in its entirety for all purposes.
[0058] Examples of methods and apparatus for signal detection and
processing of intensity data are disclosed in, for example, U.S.
Pat. Nos. 5,143,854, 5,547,839, 5,578,832, 5,631,734, 5,800,992,
5,834,758; 5,856,092, 5,902,723, 5,936,324, 5,981,956, 6,025,601,
6,090,555, 6,141,096, 6,185,030, 6,201,639; 6,218,803; and
6,225,625, in U.S. Patent application 60/364,731 and in PCT
Application PCT/US99/06097 (published as WO99/47964), each of which
also is hereby incorporated by reference in its entirety for all
purposes.
[0059] The practice of the present invention may also employ
conventional biology methods, software and systems. Computer
software products of the invention typically include computer
readable medium having computer-executable instructions for
performing the logic steps of the method of the invention. Suitable
computer readable medium include floppy disk, CD-ROM/DVD/DVD-ROM,
hard-disk drive, flash memory, ROM/RAM, magnetic tapes and etc. The
computer executable instructions may be written in a suitable
computer language or combination of several languages. Basic
computational biology methods are described in, e.g. Setubal and
Meidanis et al., Introduction to Computational Biology Methods (PWS
Publishing Company, Boston, 1997); Salzberg, Searles, Kasif, (Ed.),
Computational Methods in Molecular Biology, (Elsevier, Amsterdam,
1998); Rashidi and Buehler, Bioinformatics Basics: Application in
Biological Science and Medicine (CRC Press, London, 2000) and
Ouelette and Bzevansi Bioinformatics: A Practical Guide for
Analysis of Gene and Proteins (Wiley & Sons, Inc., 2.sup.nd
ed., 2001).
[0060] The present invention also makes use of various computer
program products and software for a variety of purposes, such as
probe design, management of data, analysis, and instrument
operation. See, for example, U.S. Pat. Nos. 5,593,839, 5,795,716,
5,733,729, 5,974,164, 6,066,454, 6,090,555, 6,185,561, 6,188,783,
6,223,127, 6,229,911 and 6,308,170.
[0061] Additionally, the present invention may have preferred
embodiments that include methods for providing genetic information
over networks such as the Internet as shown in, for example, U.S.
patent application Ser. No. 10/063,559, 60/349,546, 60/376,003,
60/394,574, 60/403,381.
[0062] Throughout this specification, various aspects of this
invention are presented in a range format. It should be understood
that the description in range format is merely for convenience and
brevity and should not be construed as an inflexible limitation on
the scope of the invention. Accordingly, the description of a range
should be considered to have specifically disclosed all the
possible subranges as well as individual numerical values within
that range. For example, description of a range such as from 1 to 6
should be considered to have specifically disclosed subranges such
as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6,
from 3 to 6 etc., as well as individual numbers within that range,
for example, 1, 2, 3, 4, 5, and 6. This applies regardless of the
breadth of the range. In addition, the fractional ranges are also
included in the exemplified amounts that are described. Therefore,
for example, a range between 1-3 includes fractions such as 1.1,
1.2, 1.3, 1.4, 1.5, 1.6, etc.
[0063] In other embodiments the activation status of the PI3K
pathway is determined using one or more gene expression products of
one or more biomarkers of the PI3K pathway. Said gene expression
products may be nucleotide or amino acid products and can be
detected using methods known in the art.
[0064] In some embodiments of the invention the activation status
of the PI3K pathway is determined by assessing the activation of
IGF1R, wherein activation of IGF1R is indicative of activation of
the PI3K pathway. In other embodiments of the invention the
activation status of the PI3K pathway is determined by assessing
the activation of PKC, wherein activation of PKC is indicative of
activation of the PI3K pathway. In some embodiments of the
invention the activation status of the PI3K pathway is determined
by assessing the expression of one or more biomarkers for the PI3K
pathway disclosed in [29], the teachings of which are incorporated
herein by reference.
[0065] In one embodiment the activation status of the Np63 pathway
is determined using gene expression data for one or more biomarkers
of the Np63 pathway. For example, in some embodiments at least one
of said one or more biomarkers is a gene which is increased upon
Np63 activation, and in other embodiments at least one of said one
or more biomarkers is a gene which is decreased upon Np63
activation. Combinations of biomarkers which are increased and
decreased upon Np63 activation may also be used. In particular
embodiments at least one of said one or more biomarkers is a gene
which is upstream of Np63 activation, while in other embodiments at
least one of said one or more biomarkers is a gene which is
downstream of Np63 activation.
[0066] In particular embodiments, expression data for said one or
more biomarkers of the Np63 pathway is obtained using an
oligonucleotide microarray. In other embodiments the activation
status of the Np63 pathway is determined using one or more gene
expression products of one or more biomarkers of the Np63 pathway.
Said gene expression products may be nucleotide or amino acid
products and can be detected using methods known in the art.
[0067] The invention also relates to a method of identifying an
individual at increased risk of lung disease, comprising
determining the activation status of PKC in a cytologically normal
airway epithelial cell from said individual, wherein activation of
PKC is indicative that said individual is at increased risk of lung
disease as compared with an individual in whom PKC is not
activated.
[0068] The invention further relates to a method of identifying an
individual at increased risk of lung disease, comprising
determining the activation status of IGF1R in a cytologically
normal airway epithelial cell from said individual, wherein
activation of IGF1R is indicative that said individual is at
increased risk of lung disease as compared with an individual in
whom IGF1R is not activated.
[0069] In other embodiments the invention provides an
oligonucleotide array having immobilized thereon one or more probes
for one or more biomarkers of the PI3K pathway, and wherein said
array does not have immobilized thereon probes for other
biomarkers. In preferred embodiments said one or more biomarkers of
the PI3K pathway are selected from the group consisting of IGF1R,
PKC, the biomarkers disclosed in [29], and combinations
thereof.
[0070] The invention also relates to a method of reducing the risk
of lung disease in an individual comprising administering to an
individual at risk of lung disease one or more agents (e.g., one or
more agents, regimens or treatments or combinations thereof) which
inhibit the PI3K pathway. In particular embodiments the PI3K
pathway is activated in said individual prior to administration of
said one or more agents. In one embodiment the lung disease is lung
cancer. In another embodiment said one or more agents are
administered to said individual prophylactically before the
development of lung disease.
[0071] The practice of the present invention will employ, unless
otherwise indicated, conventional techniques of molecular biology
(including recombinant techniques), microbiology, cell biology,
biochemistry, nucleic acid chemistry, and immunology, which are
well known to those skilled in the art. Such techniques are
explained fully in the literature, such as, Molecular Cloning: A
Laboratory Manual, second edition (Sambrook et al., 1989) and
Molecular Cloning: A Laboratory Manual, third edition (Sambrook and
Russel, 2001), (jointly referred to herein as "Sambrook"); Current
Protocols in Molecular Biology (F. M. Ausubel et al., eds., 1987,
including supplements through 2001); PCR: The Polymerase Chain
Reaction, (Mullis et al., eds., 1994); Harlow and Lane (1988)
Antibodies, A Laboratory Manual, Cold Spring Harbor Publications,
New York; Harlow and Lane (1999) Using Antibodies: A Laboratory
Manual Cold Spring Harbor Laboratory Press, Cold Spring Harbor,
N.Y. (jointly referred to herein as "Harlow and Lane"), and
Beaucage et al. eds., Current Protocols in Nucleic Acid Chemistry
John Wiley & Sons, Inc., New York, 2000).
[0072] The teachings of all references and websites cited herein
are incorporated herein by reference in their entirety. The
invention will be further described by the following non-limiting
exemplary embodiment.
Exemplary Embodiment
[0073] Methods
[0074] Patient Population
[0075] Airway epithelial brushings were collected from current and
former smokers under suspicion of lung cancer who were undergoing
diagnostic flexible bronchoscopy from four institutions--Boston
University Medical Center, Boston Veterans Administration, Lahey
Clinic and St. James's Hospital (previously described in [7], see
demographics in Table 1 for samples used in this study). Additional
brushes were collected from volunteer healthy current, former and
never smokers, as well as smokers with COPD, who were undergoing
bronchoscopy (some previously published in [31], demographics in
Table 1 and 2). Brushings from the cytologically normal bronchial
airway of current and former smokers with airway dysplasia was
collected at the University of British Columbia from volunteers who
were between 40-74, had 30 pack-years of cumulative smoke history
and had one or more sites of bronchial dysplasia on
autofluorescence bronchoscopy (see demographics in Table 2). A
subset of these volunteers were treated with myo-inositol for a
period of 2-3 months, and an additional brush was collected from
cytologically-normal airway epithelium at the end of the treatment
when autofluorescence bronchoscopy and endobronchial biopsy was
used to measure changes in dysplasia (n=20 samples, 10 individuals)
[35].
[0076] Prospective samples used for biochemical validation were
collected at both Boston University Medical Center and University
of Utah Hospital. Cytologically-normal bronchial airway brushings
were collected on subjects undergoing bronchoscopy for clinical
suspicion of lung cancer (see Table 3). Subjects were followed
post-bronchoscopy until a final diagnosis or lung cancer or an
alternative lung pathology was made.
[0077] The study was approved by the Institutional Review Board of
all participating institutions and all subjects provided written
informed consent.
[0078] Sample Collection and Processing
[0079] Cytologically normal airway epithelial samples from smokers
with and without cancer (n=129, GSE4115), as well as current,
former and never smokers (n=104, GSE7895) and smokers with (n=127)
and without (n=20) COPD (subset of GSE4115, GSE7895 and GSEYYYY)
were collected and hybridized onto Affymetrix HG-U133A microarrays
as previously described [7, 31]. For lung tumor and adjacent normal
studies, GSE10072 was used [32].
[0080] Cytologically normal airway epithelial samples from patients
with dysplastic airway lesions at the University of British
Columbia were collected before and after 2-3 months of treatment
with myo-inositol (n=20, 10 patients with two samples each), as
well as six additional samples collected pre-treatment with
myo-inositol as part of a dose response study. In that study,
bronchial brushing was performed in three separate 6-8th generation
bronchial airways using a 1.7 mm diameter bronchial cytology brush
(Hobbs Medical, Stafford Springs, Conn.). The brush was retrieved
and immediately immersed in RNALater and kept frozen at -80.degree.
C. until assayed. Epithelial cell content of representative
bronchial brushing samples has been quantitated by
cytocentrifugation (ThermoShandon Cytospin, Pittsburgh, Pa.) of the
cell pellet and staining with a cytokeratin antibody (Signet,
Dedham Mass.). The cells in the bronchial brush contained >90%
bronchial epithelial cells. At least 1 .mu.g of each sample were
later hybridized to Affymetrix Human Exon ST microarrays according
to the manufacturer's protocol. Data from exon arrays were
normalized using RMA-sketch in the Affymetrix Expression Console
software.
[0081] Airway samples collected prospectively for biochemical
validation were snap frozen in liquid nitrogen. For a subset of
patients, additional brushes were collected and hybridized to
Affymetrix HG U133A 2.0 chips. Microarrays were MAS5.0 normalized
in Affymetrix Expression Console.
[0082] All new expression data for this study is available for
download at GEO under the accession GSEYYYY.
[0083] Oncogenic Pathway Activation Probability Calculation
[0084] Oncogenic pathways from a prior study were utilized, and
calculated as detailed previously [13]. Briefly, primary mammary
epithelial cells were cultured and allowed to grow to quiescence.
Cell cultures were then infected with adenovirus constructs for key
members of a specific pathway (e.g., p110, the catalytic subunit of
PI3K) in order to activate the pathway of interest. Samples from
the perturbed and normal cell culture were processed and hybridized
onto Affymetrix microarrays in replicate (approximately 10 samples
for each pathway) 18 hours after infection. Before statistical
analysis, probesets were filtered based on low expression or low
variance (lowest 25% of each was removed). A gene signature for
each pathway was defined by selecting 200 probesets based on
correlation with the class variable (e.g., perturbed vs. GFP
control). Training of the metagene model was accomplished using the
perturbed and GFP control samples, by first summarizing the pathway
signature in the training data using the most dominant component
from singular value decomposition (SVD), and then using Bayesian
fitting of a probit regression model. This was done for each of the
pathways, and each model was applied to samples of interest.
Resulting pathway probabilities were scaled between zero and one.
To determine whether an oncogenic pathway was differentially
activated, first a rank-sum test was performed, and for p-values
less than 0.05, a random permutation analysis was performed. During
random permutation, gene identifiers in the dataset of interest
(e.g., bronchial airway) were randomized, and a p-value from a
Wilcoxon rank sum test was calculated to measure differential
activation between class variables (e.g., lung cancer vs. no lung
cancer). This was repeated 1,000 times.
[0085] Microarray data was initially preprocessed by RMA
normalizing, and then corrected for batch effects using DWD [43].
Specifically, to standardize expression data in the development of
metagene models, DWD was applied to correct batch effects between
the oncogenic pathway signature microarray samples, and bronchial
airway microarray samples.
[0086] Samples in the prospective series that were run on
microarrays in order to compare predicted PI3K activity to
biochemical measurements were normalized by mas5 due to the small
samples size (n=4). Affymetrix U133A 2.0 chips were used. Pathway
activity was calculated using MAS5.0 normalized oncogenic pathway
signatures, and no DWD standardization was utilized.
[0087] Differences between the metagene model used in this
manuscript over the original framework are as follows. Previously,
to standardize the training dataset with the samples of interest,
singular value decomposition was analyzed across all samples, and
the two most dominant components were used in the model. The first
component generally explained the variance caused by batch effects
between the signature dataset and the dataset of interest, while
the second component explained the pathway of interest. To remove
the standardization from the model, which also removes influence of
the test dataset when training the model, batch correction was done
in advance (e.g., using DWD), and SVD was applied to the training
and test datasets separately (once for the training set to train
the model, and a second time for the dataset of interest to apply
the model).
[0088] GSEA
[0089] Gene Set Enrichment Analysis [30] was calculated using GSEA
v2. The genes making up the oncogenic pathway signature for PI3K
were used to define a PI3K gene set. Importantly, two gene sets
were created, one with genes that are increased upon PI3K
activation, and one with genes that are decreased upon PI3K
activation. Three different analyses were conducted with GSEA, two
of which used microarray data from Affymetrix exon arrays. First,
healthy current smokers (n=11) were compared to smokers with
dysplasia (n=14). Genes were ranked using the following linear
model (as calculated in R):
Y=.beta..sub.0+.beta..sub.1+.beta..sub.2+b+.epsilon.
[0090] Where Y is the expression of a gene, .beta..sub.0 is the
intercept, .beta..sub.1 measures lung cancer risk (whether the
sample has dysplasia or not), .beta..sub.2 is the cumulative
cigarette smoke exposure for each person (pack-years), b is a
random effect correcting for batch differences, and .epsilon. is
the error term. The coefficient of .beta..sub.1 was used to rank
the genes for GSEA. Second, to compare pre- and post-treatment with
myo-inositol, samples were ranked using a paired Wilcoxon rank sum
test. The GSEA analysis done in the U133A airway dataset used the
default signal to noise ranking. 100 gene set permutations were
used to calculate FDR.
[0091] Kinase Assay
[0092] 80% confluent BEAS-2B, BT549, or HEK293 cells were starved
in BEBM, RPMI, or DMEM medium, respectively (Clonetics, GibcoBRL).
This media contained either 0.1% added supplements for the BEAS-2B
cells or 0.1% fetal bovine serum for BT549 and HEK293 cells for 24
hrs. Cells were then pretreated with increased concentrations of
myo-inositol (Sigma), or LY294002 (Sigma) for 16 hrs. at 37.degree.
C. Prior stimulation, the cells were treated with fresh drugs for
another 30 min, then 500 uM of insulin (SIGMA) were added for 15
minutes at 37.degree. C. The cells were lysed in RIPA buffer (20 mM
TRIS (pH 7.4), 150 mM NaCl, 1% NP-40, 0.5% Sodium Deoxycholate, 1
mM EDTA, 0.1% SDS) containing 0.1 mM sodium orthovanadate, 2 mM
PMSF, 100 uM protease inhibitors (Sigma). Lysates were centrifuged
at 14000 rpm for 20 minutes at 4.degree. C. and incubated with
monoclonal anti-p85 PI3K (Santa Cruz) antibody for 1 hr at
4.degree. C. The bounded proteins were precipitated with 50 ul of
50% slurry protein G Sepharose (Sigma) and washed three times with
lysis buffer, three times with buffer containing 0.1 mM Tris (pH
7.4), 5 mM LiCl, 0.1 mM sodium orthovanadate, and two times with
buffer containing 10 mM Tris (pH 7.4), 150 mM NaCl, 5 mM EDTA, 0.1
mM sodium orthovanadate. The beads were washed in kinase buffer (50
mM Tris (pH 7.4), 10 mM MgCl.sub.2) containing 20 uM cold ATP
(Sigma), and resuspended in 45 ul of kinase buffer containing 5 ul
of L-a-phosphatidylinositol-4,5-bisphosphate (Avanti Polar Lipids)
(1 mg/ml), and 20 uCi ATP (32-P) for 20 minutes at RT. The
reactions were stopped by addition of 100 ul 1N HCl, and the lipids
were extracted with 160 ul of CHCl3/MeOH (1:1). The phosphorylated
products were separated by TLC on Silica 60 plates pretreated with
potassium oxalate in a CHCl3/MeOH/NH4 solution (45:35:1.5). The
production of PIP3 was evaluated by autoradiography and quantified
by densitometry analysis and scintillation analysis. All
experiments for each cell line were repeated at least twice with
similar results.
[0093] Western Blot Analysis
[0094] Patient tissue samples were collected by bronchoscopy and
snap frozen immediately in liquid nitrogen. Cell extracts from
bronchoscopy brushes were prepared by adding 200 ul of RIPA buffer.
To facilitate the detachment of the cells from the brushes, the
tubes were vortexed three times for 5 seconds. Both cell and
bronchoscopy brushing extracts were centrifuged at 14000 rpm for 20
minutes at 4.degree. C. and the pellets discarded. The protein
yield was quantified by Bradford assay, and equivalent amount of
protein was loaded to 7% SDS-PAGE gels. The membrane were blocked
for 1 h in blocking buffer (Tris buffer saline containing 0.1%
Tween 20 and 2.5% BSA, or Tris buffer saline containing 0.1% of
Tween 20 and 5% low fat milk), and placed in primary antibody (Tris
buffer saline containing 0.1% Tween 20 and 2.5% BSA, 0.02% sodium
azide) overnight at 4.degree. C. The primary antibodies used in
this study are the followed: rabbit phospho-PKC (pan) (pII Ser660)
ratio 1:100 (Cell Signaling Techn.); rabbit phospho-IGF-I Receptor
.beta. (Tyr1131)/Insulin Receptor .beta. (Tyr 1146) ratio 1:100
(Cell Signaling Techn.); rabbit phospho-PLCy1 (Tyr783), ratio 1:500
(Cell Signaling Techn.); rabbit phospho-AKT (Ser473) ratio 1:100
(Cell Signaling Techn.); goat PI3-Kinase p110.alpha. (C17) ratio
1:50 (Santa Cruz), rabbit phospho-ERK ratio 1:100 (Cell Signaling)
rabbit GAPDH, ratio 1:1000 (AbCam). Nitrocellulose were washed
three times in Tris buffer saline containing 0.1% Tween 20 and/or
0.1% NP-40. Primary antibody was detected using horseradish
peroxidase-linked secondary antibody and visualized with the ECL
Plus Western Blot Detection system (GE Healthcare).
[0095] Results
[0096] PI3K Pathway Activation in Cytologically Normal Bronchial
Airway Epithelial Cells of Smokers with Lung Cancer
[0097] Cytologically normal bronchial airway epithelial cell
brushings were obtained from current and former smokers undergoing
flexible bronchoscopy for suspicion of lung cancer and were
hybridized to DNA microarrays as previously described [7] (n=129,
see patient demographics in Table 1). To help elucidate oncogenic
pathway signaling changes in these cells, we utilized a previously
published gene expression dataset and computational approach [13,
26-28]. Oncogenic pathway signatures [13] were experimentally
derived by activating a pathway via expression of a specific
oncogene in primary human epithelial cells. A gene expression
signature was then defined by identifying which genes are altered
following pathway activation, and used to predict pathway activity
in other in vivo samples. Using this methodology, oncogenic pathway
activation probabilities for seven signaling pathways (Ras, Myc,
E2F3, Src, .beta.-catenin, .DELTA.Np63 and phosphatidylinositol 3'
kinase (PI3K)) were calculated for the bronchial airway epithelial
of current and former smokers with suspicion for lung cancer[7]. It
is important to note that although approximately half of these
patients were ultimately diagnosed with primary lung cancer (the
remainder were found to have alternate lung pathologies), the
brushings collected from the proximal mainstem bronchus (i.e. not
adjacent to the tumor or lung lesion), were cytologically normal
and were >90% epithelial. Thus, a priori one would not expect
differential oncogenic pathway activity in the normal airway of
smokers with lung cancer.
[0098] Of the seven pathways tested, only two were found to be
significantly and differentially activated in the airway of smokers
with lung cancer compared to controls with alternate lung
pathologies after a random permutation analysis: .DELTA.Np63 and
PI3K (p<0.001, FIG. 1A). Furthermore, the genes that have been
found to play roles in the phosphatidylinositol signaling system
pathway [29, the teachings of which are incorporated by reference
herein] were also found to be significantly upregulated in lung
cancer patients using Gene Set Enrichment Analysis [30] (GSEA,
p=0.034, FDR q=0.099). Upregulation of PI3K was not limited to a
specific cancer cell type, tumor location or tumor stage (data not
shown).
[0099] PI3K Activation is not Significantly Correlated to
Cumulative Smoke Exposure or COPD
[0100] We next sought to determine whether the increased pathway
activity of PI3K was due to the presence of cancer in the lung, or
caused by other confounding factors such as differences in
cumulative smoke exposure (patients with cancer had higher
cumulative exposure, see Table 1) or other pulmonary diseases.
First, an ANCOVA using cumulative smoke exposure as a covariate was
used to test for differential pathway activation between patients
with and without lung cancer. PI3K (p=2.08.times.10-8) remained
significantly differentially activated after addressing the
possible confounding influence of differences in tobacco exposure.
Second, using a whole-genome gene expression dataset of bronchial
airway epithelium collected from current (n=52), former (n=31) and
never (n=21) healthy smokers [31], we calculated the pathway
activation probabilities for all seven pathways using the same
methodologies previously described. While none of the seven
pathways were differentially activated between healthy current,
never and former smokers, the .DELTA.Np63 pathway trended towards
being significantly activated when comparing never and current
smokers (p=0.09, FIG. 1B). Finally, using a bronchial airway gene
expression dataset obtained from smokers with (n=17) and without
(n=20) chronic obstructive pulmonary disease (COPD), Ras was the
only pathway that was differentially activated (p<0.001, data
not shown) (FIG. 10). This lends evidence that the significant
differential activation of the oncogenic pathway PI3K in the normal
bronchial airway is specific to individuals with lung cancer,
though the mechanism of differential activation is unknown. Due to
the known deregulation of p53 related pathways (such as
.DELTA.Np63) in response to environmental stressors, as well as the
lack of any known therapeutic modulators of the p53/p63 pathway, we
chose to focus on the PI3K pathway for all further studies.
[0101] PI3K is Activated in Lung Cancer Tissue
[0102] The deregulation of PI3K in cytologically normal airway
epithelial cells of patients with lung cancer led us to examine
PI3K activity in lung cancer tissue. We would expect a significant
increase in oncogenic pathways essential for tumor growth and
survival during lung cancer development. For this analysis, we used
a published dataset comprised of lung adenocarcinoma and matched
adjacent non-tumor tissue (n=107) [32]. Pathway status was
predicted using the same genomic approach detailed above. As shown
in FIG. 2, malignant lung tumors had a highly significant
(p<0.001) increase in PI3K activity as compared to the adjacent
non-tumor tissue. .DELTA.Np63 pathway was also increased to a
lesser extent in tumor cells (p=0.002). This result highlights a
central role for the PI3K pathway in the malignant progression of
cells, and supports our hypothesis that PI3K activation is
important for lung cancer tumorigenesis. A significant increase was
also seen for the Myc, E2F3 and Src pathways in lung tumors,
confirming their roles in lung tumor growth and development. As
lung tumors are comprised of dividing cells and have higher levels
of proliferation than normal tissue, we would expect pathway
involved in cell growth (Myc, E2F3, Src) to also be increased in
this analysis.
[0103] Biochemical Analysis of PI3K Pathway Activity in the
Bronchial Airway
[0104] To validate our gene expression findings, we measured PI3K
enzymatic activity in a prospectively collected cohort of
cytologically-normal airway epithelial samples from subjects
undergoing bronchoscopy for clinical suspicion of lung cancer.
Samples were independently obtained from Boston University Medical
Center and University of Utah Hospital between October 2007 and
June 2008. Subjects were followed post-bronchoscopy until a final
diagnosis of lung cancer or an alternate lung pathology was made.
Importantly, subjects without lung cancer had a range of other
pathologies, including metastatic cancer of non-lung origin,
sarcoidosis, septic emboli, and pneumonia. Following protein
extraction from the airway brushings, a PI3K kinase assay was
performed. Based on our genomic predictions, we would expect a
majority of samples from patients with cancer to have high PI3K
activity and only a minority of the samples from patients with
alternative pathologies to have high PI3K activity.
[0105] As seen in FIG. 3A, PI3K showed increased activation in the
majority of patients with lung cancer (70% of the lung cancer
samples in the genomic analysis were in the top half of PI3K
activity), as compared to patients without lung cancer (30%).
Specifically, we see high correlation between PI3K activity and
lung cancer status for both cohorts (Boston: R=0.499, Utah:
R=0.389). For a subset of these samples (n=4), we were able to
collect additional bronchial epithelial cells to perform microarray
analysis and predict PI3K activity using the same approach as was
used on the original dataset. Predicted PI3K activity was
correlated with the PI3K levels measured biochemically within the
same individual (R=0.48). Together, these results support the
conclusion that PI3K activity is increased in the normal airway of
patients with lung cancer and validate our computational
predictions of PI3K activity calculated from gene expression data.
These results are even more striking when considering the variety
of pathologies in the control patients that might also affect PI3K
activity.
[0106] In an attempt to further define the causes and consequences
of elevated PI3K activity, we performed Western Blotting analysis
of the phosphorylation state of key PI3K pathway components.
Phosphorylation of IGF1R (Boston: R=0.91, Utah: R=0.34), but not
HER2 (Boston R=-0.61, Utah R=-0.41), positively correlated to PI3K
kinase activity (FIG. 3B), suggestive of this receptor being an
upstream effector for PI3K activation in the normal airway
epithelial tissue of patients with lung cancer. Correlation between
biochemically measured PI3K activity and IGF1R in patients with
lung cancer shows a high positive correlation, further supporting a
potential relationship between IGF1R activation and PI3K activity
(Boston R=0.98, Utah R=0.90) (FIG. 3B). Next, measured
phosphorylation of Akt and PKC, the major downstream effectors of
PI3K, was measured. PKC had higher correlation with biochemically
measured PI3K activity in patients with lung cancer, while no
significant correlation was seen for Akt (FIG. 3B), again
highlighting activation of specific PI3K pathways in patients with
lung cancer. Cumulatively, these results support our genomic
findings that PI3K activity is enriched in the normal lung cells of
patients with lung cancer compared to patients with other
pathologies, and the activation of IGF1R is a potential mechanism
for this activity [33, 34].
[0107] PI3K Pathway Activation in High-Risk Smokers with
Dysplasia
[0108] To evaluate the hypothesis that an increased activation of
PI3K is an early event in the development of lung cancer, we
compared gene expression in cytologically normal airway epithelia
from a group of healthy smokers (n=11) to a group of smokers with
moderate-severe airway dysplasia (n=14, from [35]) (See Table 2).
As dysplasia is considered to be a pre-neoplastic event, this
cohort represents "high-risk" smokers who have an increased
likelihood of getting lung cancer [36, 37]. If high-risk smokers
with dysplasia have higher levels of PI3K than healthy smokers, it
would suggest that the increased activity in the airway epithelium
precede the development of lung cancer. The high-risk cohort, as
well as a cohort of healthy current smokers, was hybridized to
Affymetrix Human Exon arrays. Due to platform differences with the
oncogenic pathway signatures, we were unable to use the metagene
model to calculate pathway activity. Instead, GSEA was used to
compare PI3K activity between the two groups, and the PI3K gene
signature defined by the in vitro experiments was split into two
gene sets defined by the genes that go up or down with PI3K
activation (PI3K_Up, PI3K_Down). Given that there were significant
differences in cumulative tobacco exposure between the two groups,
a linear model incorporating both batch effects and pack-years was
used to rank the genes for use in GSEA. A significant increase in
PI3K activity in the cytologically normal airway of subjects with
dysplasia was found (p<0.001, FDR Q=0.022 and p<0.001,
Q<0.001 for PI3K_Up and PI3K_Down gene sets respectively) (FIG.
4). The increased activity of PI3K in the airway of people with
dysplasia indicates that the deregulation of PI3K is an early event
in lung cancer tumorigenesis.
[0109] Reversibility of PI3K Activity in High-Risk Smokers Treated
with Myo-Inositol
[0110] Given elevated PI3K activity in both high-risk smokers as
well as smokers with lung cancer, we next wanted to determine if a
decrease of PI3K activity would correlate with regression of
dysplastic lesions. A recent phase 1 clinical study published by
Lam et al. [35] studied myo-inositol as a lung cancer
chemoprophylactic agent in high-risk smokers. In this study,
volunteer smokers with 30 pack-years of smoking history were
screened for the presence of dysplasia in their airway using
autofluorescent bronchoscopy. Ten current and former smokers with
moderate-severe dysplasia documented on endobronchial biopsy were
then given oral myo-inositol for 2-3 months, and the status of
their dysplastic lesions was again measured by repeat endobronchial
biopsy of the site. When compared to control patients who were
treated with placebo, myo-inositol was found to significantly
increase the rate of dysplastic regression. Airway brushings of
cytologically normal bronchial epithelium were also collected both
before and after treatment, and gene expression profiling was
performed (see demographics in Table 2).
[0111] GSEA analysis was again used to measure changes in the
expression of genes in the PI3K pathway. When comparing before
treatment with myo-inositol versus after treatment, subjects who
did respond to treatment (n=6, 12 samples) showed increased
expression of genes in the PI3K Down gene set (p=0.04, FDR
Q=0.177), which is suggestive of a decrease in PI3K levels
following treatment with myo-inositol. Those that did not respond
to treatment (n=3, 6 samples) had no change in the levels of the
PI3K gene sets. The decrease in airway PI3K activity seen in
patients who respond to myo-inositol demonstrates that regression
of dysplasia is correlated to the activity level of this
pathway.
[0112] Myo-Inositol as a PI3K Inhibitor
[0113] A reduction in PI3K activity in patients that responded to
myo-inositol with a regression of dysplasia in their airway
reinforces the association between elevated PI3K activity levels
and the presence of pre-neoplastic airway lesions. However, the
mechanism of action of myo-inositol has remained undefined, and the
study was limited by a relatively small sample size. To further
explore the relationship between myo-inositol and the PI3K
activity, we tested the ability of myo-inositol to inhibit PI3K in
vitro. Following activation of PI3K by treating cells with insulin,
cells were treated with increasing doses of either myo-inositol or
LY-294002 (a known PI3K inhibitor). PI3K activity, as measured by
PIP3 levels, was then quantified using a standard kinase assay
protocol [38, 39]. For this experiment, three different cell lines
were analyzed in replicate: BEAS-2B (airway), BT549 (breast cancer)
and HEK293 (embryonic kidney). In all three cell lines,
myo-inositol inhibited PI3K activity levels in a dose-dependent
manner (FIG. 5). Thus, myo-inositol is an inhibitor of PI3K, and
has chemoprophylactic properties associated with regression of
dysplasia in airway epithelial cells.
[0114] Tables:
TABLE-US-00001 TABLE 1 Patient demographics for the Affymetrix
U133A platform datasets used in this study. These cohorts were used
to explore differences in oncogenic activity in the cytologically
normal airway epithelium of smokers with lung cancer. Average
number for clinical variables is shown, with the standard deviation
in parenthesis. Under "Smoking Status," C = current smoker, and F =
Former smoker. COPD = Chronic Obstructive Pulmonary Disease. All
datasets consist of samples collected via brushing of
cytologically-normal bronchial airway epithelium obtained during
bronchoscopy. Primary Dataset Never/Former/Current Smoker Dataset
COPD Dataset Luna Cancer No Lung Cancer Never Former Current No
COPD COPD Samples 60 69 21 31 52 20 17 Age 64.1 (9)* 49.8 (15.2)*
32.3 (10.7)* 55.9 (14.7)* 48.6 (15.2)* 43.6 (9.3)* 60.4 (12.3)*
Pyers 57.4 (25.6)* 29.4 (27.3)* 34.0 (30.1) 34.5 (34.2) 33.5
(25.9)* 57.4 (27.9)* Smoking Status 51.7% F, 48.3% C 37.3% F, 62.3%
C 100% F 100% C 100% C 100% C Months Since Quit 113 (118) 158 (159)
145.2 (162.8) (Formers) *Feature is significantly different between
the classes of that dataset (p < 0.05)
TABLE-US-00002 TABLE 2 Patient demographics for the Affymetrix exon
array platform datasets used in this study. These cohorts were used
to explore PI3K activity prior to the development of lung cancer,
as well as changes in PI3K activity following treatment with a lung
cancer chemoprophylaxis agent (myo-inositol). Average number for
clinical variables is shown, with the standard deviation in
parenthesis. Under "Smoking Status," C = current smoker, and F =
former smoker. All datasets consist of samples collected via
brushing of cytologically-normal bronchial airway epithelium
obtained during bronchoscopy. Smokers With Dysplasia (Myo-inositol
dataset) Matched Samples Before Airway with Healthy Smokers and
After Treatment Dysplasia Current Samples 20 (10 before, 10 after)
6 11 Age 61.7 (8.3) 64.5 (7.1) 36.5 (10.0) Pack-Years 42.9 (23.7)
59.8 (17.7) 12.4 (9.2) Smoking Status 70% F, 30% C 100% C 100% C
Months Since Quit (Formers) 145.7 (89.9) * Age, Pack-years
significantly different between dysplasia and healthy smoker
datasets (p < 0.05)
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