U.S. patent application number 15/313301 was filed with the patent office on 2017-07-06 for non-invasive gene mutation detection in lung cancer patients.
The applicant listed for this patent is THE REGENTS OF THE UNIVERSITY OF CALIFORNIA. Invention is credited to Wu-Chou Su, Fang Wei, David T.W. Wong.
Application Number | 20170191118 15/313301 |
Document ID | / |
Family ID | 54767327 |
Filed Date | 2017-07-06 |
United States Patent
Application |
20170191118 |
Kind Code |
A1 |
Wei; Fang ; et al. |
July 6, 2017 |
Non-Invasive Gene Mutation Detection in Lung Cancer Patients
Abstract
A system and method for the detection of saliva biomarkers in
bodily fluids is described. In particular, the system is suitable
for detecting biomarkers of lung cancer in a subject. The system
includes an electrochemical sensor chip having at least one well,
wherein the at least one well contains a working electrode coated
with a conducting polymer functionalized with at least one capture
probe, and at least one labeled detector probe. When the at least
one labeled detector probe is mixed with a sample of the subject
containing a biomarker of lung cancer and added to the at least one
well, an electric current is applied to the sample, such that when
at least some of the biomarker binds to the capture probe, a
measurable change in electric current in the sample is created that
is indicative of lung cancer.
Inventors: |
Wei; Fang; (North Hills,
CA) ; Wong; David T.W.; (Beverly Hills, CA) ;
Su; Wu-Chou; (Tainan, TW) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
THE REGENTS OF THE UNIVERSITY OF CALIFORNIA |
Oakland |
CA |
US |
|
|
Family ID: |
54767327 |
Appl. No.: |
15/313301 |
Filed: |
June 3, 2015 |
PCT Filed: |
June 3, 2015 |
PCT NO: |
PCT/US2015/034028 |
371 Date: |
November 22, 2016 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
62007286 |
Jun 3, 2014 |
|
|
|
Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C12Q 2600/156 20130101;
G01N 27/3276 20130101; G01N 33/5438 20130101; G01N 33/57423
20130101; C12Q 1/6886 20130101; C12Q 1/6825 20130101; C12Q 2600/158
20130101 |
International
Class: |
C12Q 1/68 20060101
C12Q001/68; G01N 33/574 20060101 G01N033/574; G01N 27/327 20060101
G01N027/327; G01N 33/543 20060101 G01N033/543 |
Goverment Interests
STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT
[0002] This invention was made with Government support under
TR00124, awarded by the National Institutes of Health. The
Government has certain rights in the invention.
Claims
1. A device for detecting lung cancer in a subject, comprising: an
array of units on a substrate, each unit comprising an electrode
chip including a working electrode, a counter electrode, and a
reference electrode; wherein the working electrode of at least one
unit is coated with a conducting polymer embedded or functionalized
with a capture probe which binds to a first marker of lung
cancer.
2. The device of claim 1, wherein the working electrode, counter
electrode, and reference electrode are comprised of a conductive
material.
3. The device of claim 1, wherein the conducting polymer comprises
pyrrole.
4. The device of claim 1, wherein the conducting polymer is
electropolymerized on the working electrode by applying a cyclic
square-wave electric field to the device.
5. The device of claim 1, wherein the first marker of lung cancer
is a nucleic acid.
6. The device of claim 1, wherein the capture probe is a nucleic
acid that hybridizes to a nucleic acid sequence encoding mutant
EGFR in a sample.
7. The device of claim 6, wherein the nucleic acid encoding mutant
EGFR encodes a EGFR mutant selected from the group consisting of
E746-A750 deletion mutant of EGFR and L858R point mutation of
EGFR.
8. The device of claim 1, wherein the capture probe is a nucleic
acid that hybridizes to a nucleic acid sequence encoding wild-type
EGFR in a sample.
9. The device of claim 1, wherein the capture probe comprises a
nucleotide sequence selected from the group consisting of SEQ ID
NO: 1 and SEQ ID NO: 3.
10. A method of detecting lung cancer in a subject comprising:
obtaining a sample of the subject; mixing a first portion of the
sample with a solution comprising a labeled detector probe; adding
the mixture to a first electrode chip on a device, the electrode
chip comprising a working electrode, a counter electrode, and a
reference electrode; wherein the working electrode is coated with a
conducting polymer embedded with a capture probe capable of binding
to first marker associated with lung cancer in the sample; and
measuring the current in the electrode chip, wherein a change in
current is correlated to the presence of the first marker
associated with lung cancer in the sample.
11. The method of claim 10, further comprising applying a cyclic
square-wave electric field to the electrode chip during the adding
step.
12. The method of claim 10, wherein the first marker comprises a
variable region comprising a nucleic acid sequence harboring a
mutation associated with lung cancer, and at least one of the
detector probe and capture probe hybridizes to the variable
region.
13. The method of claim 10, wherein the first marker associated
with lung cancer comprises a nucleic acid encoding mutant EGFR.
14. The method of claim 13, wherein the nucleic acid encoding
mutant EGFR encodes a EGFR mutant selected from the group
consisting of E746-A750 deletion mutant of EGFR and L858R point
mutation of EGFR.
15. The method of claim 13, wherein the capture probe comprises a
nucleotide sequence selected from the group consisting of SEQ ID
NO: 1 and SEQ ID NO: 3.
16. The method of claim 13, wherein the labeled detector probe
comprises a nucleotide sequence selected from the group consisting
of SEQ ID NO: 2 and SEQ ID NO: 4.
17. The method of claim 13, wherein the sample is a saliva
sample.
18. The method of claim 13, wherein the sample is a blood
sample.
19. A system for detecting lung cancer in a subject, comprising: an
electrochemical sensor chip having at least one well, wherein the
at least one well contains a working electrode coated with a
conducting polymer functionalized with at least one capture probe;
and at least one labeled detector probe; wherein, when the at least
one labeled detector probe is mixed with a sample from the subject
containing a first marker of lung cancer and added to the at least
one well, an electric current is applied to the sample in the at
least one well, such that when at least some of the first marker
binds to the capture probe, a measurable change in electric current
in the sample is created that is indicative of lung cancer.
20. The system of claim 19, wherein the first marker is a nucleic
acid encoding mutant EGFR.
21. The system of claim 19, wherein the change in current in the
sample is measurable within 10 minutes after the sample has been
loaded into the well.
22. The system of claim 19, wherein the first marker comprises a
variable region comprising a nucleic acid sequence harboring a
mutation associated with lung cancer, and at least one of the
detector probe and capture probe hybridizes to the variable
region.
23. The system of claim 20, wherein the nucleic acid encoding
mutant EGFR encodes a EGFR mutant selected from the group
consisting of E746-A750 deletion mutant of EGFR and L858R point
mutation of EGFR.
24. The system of claim 19, wherein the capture probe comprises a
nucleotide sequence selected from the group consisting of SEQ ID
NO: 1 and SEQ ID NO: 3.
25. The system of claim 19, wherein the labeled detector probe
comprises a nucleotide sequence selected from the group consisting
of SEQ ID NO: 2 and SEQ ID NO: 4.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application claims priority to U.S. Provisional Patent
Application No. 62/007,286 filed Jun. 3, 2014, the contents of
which are incorporated by reference herein in their entirety.
BACKGROUND OF THE INVENTION
[0003] Lung cancer has the highest incidence of all cancers and is
the leading cause of cancer-related deaths worldwide, accounting
for 29% of all male and 26% of all female cancer deaths (Siegel R,
Naishadham D, Jemal A. Cancer statistics, 2012. CA Cancer J Clin
2012; 62: 10-29).
[0004] Recent understanding of the pathogenesis and molecular
oncology of lung cancers has contributed to the discovery of the
biological and therapeutic importance of acquired genetic
alterations in epidermal growth factor receptor (EGFR), which
encodes a pharmacologically targetable tyrosine kinase (Lynch T J,
Bell D W, Sordella R, et al. Activating mutations in the epidermal
growth factor receptor underlying responsiveness of non-small-cell
lung cancer to gefitinib. The New England journal of medicine 2004;
350: 2129-2139; Paez J G, Janne P A, Lee J C, et al. EGFR mutations
in lung cancer: correlation with clinical response to gefitinib
therapy. Science 2004; 304: 1497-1500). A deletion in exon 19
(p.E746_A750del) and a point mutation in exon 21 (p.L858R) occur
most frequently and are associated with a high degree of
responsiveness to EGFR tyrosine kinase inhibitors (TKIs). In 2009,
the first randomized clinical trial (the Iressa Pan-Asia Study)
demonstrated that, for advanced non-small-cell lung carcinoma
(NSCLC) patients carrying an activating EGFR mutation, initial
treatment with an EGFR-TKI was superior to standard platinum-based
chemotherapy (Mok T S, Wu Y L, Thongprasert S, et al. Gefitinib or
carboplatin-paclitaxel in pulmonary adenocarcinoma. The New England
journal of medicine 2009; 361: 947-957). Subsequently, one
single-arm study and three other randomized studies confirmed the
association between activating EGFR mutations and objective
response to gefitinib and/or erlotinib therapy (Maemondo M, Inoue
A, Kobayashi K, et al. Gefitinib or chemotherapy for non-small-cell
lung cancer with mutated EGFR. N Engl J Med 2010; 362: 2380-2388;
Mitsudomi T, Morita S, Yatabe Y, et al. Gefitinib versus cisplatin
plus docetaxel in patients with non-small-cell lung cancer
harbouring mutations of the epidermal growth factor receptor
(WJTOG3405): an open label, randomised phase 3 trial. Lancet Oncol
2010; 11: 121-128; Rosell R, Carcereny E, Gervais R, et al.
Erlotinib versus standard chemotherapy as first-line treatment for
European patients with advanced EGFR mutation-positive
non-small-cell lung cancer (EURTAC): a multicentre, open-label,
randomised phase 3 trial. The lancet oncology 2012; 13: 239-246;
Zhou C, Wu Y L, Chen G, et al. Erlotinib versus chemotherapy as
first-line treatment for patients with advanced EGFR
mutation-positive non-small-cell lung cancer (OPTIMAL, CTONG-0802):
a multicentre, open-label, randomised, phase 3 study. Lancet Oncol
2011; 12: 735-742).
[0005] EGFR mutation analysis is performed on tumor cells in biopsy
or cytology specimens obtained from bronchoscopy, computed
tomography (CT)-guided biopsy, surgical resection, or drainage from
malignant pleural effusions. However, most NSCLC patients suffer
from late-stage cancer and have to be subjected to the invasive
biopsy procedure in order to provide tumor tissue for EGFR mutation
testing. In addition, the progressive development of EGFR oncogene
mutations eventually leads to drug resistance. Therefore, the
initial detection and continuous monitoring of EGFR oncogenic
mutations are important for the long-term management of NSCLC
patients; this strategy enables clinicians to adjust therapeutic
strategies, improving the clinical outcome of oncogene molecular
targeted therapy.
[0006] Given that blood harbors the same genetic lesions as the
primary tumor, blood-borne biomarkers such as circulating tumor
cells (CTCs) and circulating tumor DNA are promising for the
detection of somatic mutations derived from malignant tumors
(Bidard F C, Weigelt B, Reis-Filho J S. Going with the flow: from
circulating tumor cells to DNA. Science translational medicine
2013; 5: 207ps214). Since not all of the cells identified in blood
are CTCs, nor are CTCs of all phenotypes captured by these
approaches, it remains challenging to use CTCs to detect EGFR
mutations (Wicha M S, Hayes D F. Circulating tumor cells: not all
detected cells are bad and not all bad cells are detected. Journal
of clinical oncology: official journal of the American Society of
Clinical Oncology 2011; 29: 1508-1511). DNA extraction from plasma
samples is relatively straightforward compared to CTC collection.
However, detecting circulating tumor DNA in plasma requires the use
of molecular methods such as polymerase chain reaction (PCR)-based
technology (Kimura H, Kasahara K, Kawaishi M, et al. Detection of
epidermal growth factor receptor mutations in serum as a predictor
of the response to gefitinib in patients with non-small-cell lung
cancer. Clinical cancer research: an official journal of the
American Association for Cancer Research 2006; 12: 3915-3921;
Kimura H, Suminoe M, Kasahara K, et al. Evaluation of epidermal
growth factor receptor mutation status in serum DNA as a predictor
of response to gefitinib (IRESSA). British journal of cancer 2007;
97: 778-784), high-performance liquid chromatography (Bai H, Mao L,
Wang H S, et al. Epidermal growth factor receptor mutations in
plasma DNA samples predict tumor response in Chinese patients with
stages IIIB to IV non-small-cell lung cancer. Journal of clinical
oncology: official journal of the American Society of Clinical
Oncology 2009; 27: 2653-2659), and mutant-enriched liquid chips
(Zhang H, Liu D, Li S, et al. Comparison of EGFR signaling pathway
somatic DNA mutations derived from peripheral blood and
corresponding tumor tissue of patients with advanced non-small-cell
lung cancer using liquidchip technology. The Journal of molecular
diagnostics: JMD 2013; 15: 819-826). These techniques are
complicated, technique-dependent, and time-consuming, and are
therefore limited in clinical use.
[0007] Thus, there is a need in the art for a system and method for
guiding treatments for lung cancer that are non-invasive, always
available, minimal or no sample preparation, and provide immediate
information on EGFR mutation status. The present invention
satisfies this need.
SUMMARY OF THE INVENTION
[0008] In one aspect, the present invention provides a device for
detecting lung cancer in a subject. The device comprises an array
of units on a substrate, each unit comprising an electrode chip
including a working electrode, a counter electrode, and a reference
electrode. The working electrode of at least one unit is coated
with a conducting polymer embedded or functionalized with a capture
probe which binds to a first marker of lung cancer.
[0009] In one embodiment, the working electrode, counter electrode,
and reference electrode are comprised of a conductive material. In
one embodiment, the conducting polymer comprises pyrrole. In one
embodiment, the conducting polymer is electropolymerized on the
working electrode by applying a cyclic square-wave electric field
to the device.
[0010] In one embodiment, the first marker of lung cancer is a
nucleic acid. In one embodiment, the capture probe is a nucleic
acid that hybridizes to a nucleic acid sequence encoding mutant
EGFR in a sample. In one embodiment, the capture probe is a nucleic
acid that hybridizes to a nucleic acid sequence encoding wild-type
EGFR in a sample.
[0011] In one embodiment, the nucleic acid encoding mutant EGFR
encodes a EGFR mutant selected from the group consisting of
E746-A750 deletion mutant of EGFR and L858R point mutation of EGFR.
In one embodiment, the capture probe comprises a nucleotide
sequence selected from the group consisting of SEQ ID NO: 1 and SEQ
ID NO: 3.
[0012] In one aspect, the present invention provides a method of
detecting lung cancer in a subject. The method comprises obtaining
a sample of the subject; mixing a first portion of the sample with
a solution comprising a labeled detector probe; adding the mixture
to a first electrode chip on a device, the electrode chip
comprising a working electrode, a counter electrode, and a
reference electrode; wherein the working electrode is coated with a
conducting polymer embedded with a capture probe capable of binding
to first marker associated with lung cancer in the sample; and
measuring the current in the electrode chip, wherein a change in
current is correlated to the presence of the first marker
associated with lung cancer in the sample.
[0013] In one embodiment, the method further comprises applying a
cyclic square-wave electric field to the electrode chip during the
adding step.
[0014] In one embodiment, the first marker comprises a variable
region comprising a nucleic acid sequence harboring a mutation
associated with lung cancer, and at least one of the detector probe
and capture probe hybridizes to the variable region.
[0015] In one embodiment, the first marker associated with lung
cancer comprises a nucleic acid encoding mutant EGFR. In one
embodiment, the nucleic acid encoding mutant EGFR encodes a EGFR
mutant selected from the group consisting of E746-A750 deletion
mutant of EGFR and L858R point mutation of EGFR.
[0016] In one embodiment, the capture probe comprises a nucleotide
sequence selected from the group consisting of SEQ ID NO: 1 and SEQ
ID NO: 3.
[0017] In one embodiment, the labeled detector probe comprises a
nucleotide sequence selected from the group consisting of SEQ ID
NO: 2 and SEQ ID NO: 4.
[0018] In one embodiment, the sample is a saliva sample. In one
embodiment, the sample is a blood sample.
[0019] In one aspect, the present invention provides a system for
detecting lung cancer in a subject. The system comprises an
electrochemical sensor chip having at least one well, wherein the
at least one well contains a working electrode coated with a
conducting polymer functionalized with at least one capture probe;
and at least one labeled detector probe. In one embodiment, when
the at least one labeled detector probe is mixed with a sample from
the subject containing a first marker of lung cancer and added to
the at least one well, an electric current is applied to the sample
in the at least one well, such that when at least some of the first
marker binds to the capture probe, a measurable change in electric
current in the sample is created that is indicative of lung
cancer.
[0020] In one embodiment, the first marker comprises a variable
region comprising a nucleic acid sequence harboring a mutation
associated with lung cancer, and at least one of the detector probe
and capture probe hybridizes to the variable region. In one
embodiment, the first marker is a nucleic acid encoding mutant
EGFR.
[0021] In one embodiment, the change in current in the sample is
measurable within 10 minutes after the sample has been loaded into
the well.
[0022] In one embodiment, the nucleic acid encoding mutant EGFR
encodes a EGFR mutant selected from the group consisting of
E746-A750 deletion mutant of EGFR and L858R point mutation of
EGFR.
[0023] In one embodiment, the capture probe comprises a nucleotide
sequence selected from the group consisting of SEQ ID NO: 1 and SEQ
ID NO: 3. In one embodiment, the labeled detector probe comprises a
nucleotide sequence selected from the group consisting of SEQ ID
NO: 2 and SEQ ID NO: 4.
BRIEF DESCRIPTION OF THE DRAWINGS
[0024] The following detailed description of preferred embodiments
of the invention will be better understood when read in conjunction
with the appended drawings. For the purpose of illustrating the
invention, there are shown in the drawings embodiments which are
presently preferred. It should be understood, however, that the
invention is not limited to the precise arrangements and
instrumentalities of the embodiments shown in the drawings.
[0025] FIG. 1 is a schematic of the system platform array
technology for the detection of EGFR mutations in the body fluids
of lung-cancer patients. The cyclic-square wave of electrical field
(csw E-field) was applied to release and detect the EGFR mutation.
EGFR sequences were measured on the electrochemical sensor with
pre-coated capture probe in conducting polymer. The horseradish
peroxidase (HRP) labeled reporter probe generated amperometric
signal when reaction with TMB (3,3',5,5'-tetramethylbenzidine)
substrate under -200 mV electrical field.
[0026] FIG. 2, comprising FIG. 2A through FIG. 2F, is a set of
graphs depicting the in vitro optimization of the platform for
specific human EGFR mutation detection. Cycle numbers for the
application of EFIRM for the detection of oligos carrying the (FIG.
2A) exon 19 deletion and (FIG. 2B) the L858R mutation. Targeting
oligos were dissolved in Tris-HCl buffer at a final concentration
of 1 nM. Wild-type sequences were utilized as mismatch sequences.
The TKI-sensitive EGFR mutations as Exon 19 deletion (taken from
HCC827 cells) and L858R (taken from NCI-H1975 cells) were assayed
by decreasing the ratio of targeted oncogene sequence to other
sequences. Electrochemical current readouts are listed in (FIG. 2C)
amperometric signals for Exon 19 deletion and (FIG. 2D) L858R.
Reactions were performed in triplicate using 20 ng of input DNA.
Mean and standard deviation from triplicates experiments are
provided (FIG. 2E) for Exon 19 deletion and (FIG. 2F) L858R.
[0027] FIG. 3, comprising FIG. 3A through FIG. 3G, depicts the
detection of EGFR L858R point mutation in xenografted lung cancer
mice via the system of the present invention. (FIG. 3A) Design of
the tumor burden study using EGFR L858R xenografted mice. Testing
of the four groups of mice with (FIG. 3B) 10 .mu.L of plasma
(R=0.86), (FIG. 3C) 20 .mu.L of plasma (R=0.98), and (FIG. 3D) 40
.mu.L of plasma (R=0.95). Reactions were performed in triplicate
with both the mean and standard deviations provided. Linear fits to
the data appear in red with the correlation co-efficient R
provided. Amperometric current readout are listed in (FIG. 3E) with
10 .mu.l of plasma, (FIG. 3F) 20 .mu.l of plasma and (FIG. 3G) 40
.mu.l of plasma.
[0028] FIG. 4, comprising FIG. 4A through FIG. 4E, depicts the
detection of EGFR mutations in plasma and saliva from NSCLC
patients via the system of the present invention. The platform was
compared with biopsy determined EGFR mutation type directly in
plasma and saliva from lung cancer patients. To detect the specific
mutation type, corresponding probes were applied to the
electrochemical sensor to detect the specific mutation type. (FIG.
4A) The probe for exon 19 deletion in saliva; (FIG. 4B) the probe
for L858R in saliva; (FIG. 4C) the probe for exon 19 deletion in
plasma; and (FIG. 4D) the probe for L858R in plasma.
(***P<0.0001, one-way analysis of variance and Bonferroni post
hoc test). (FIG. 4E) Amperometric current results with the probe
for exon 19 deletion in saliva from patients with lung cancer.
[0029] FIG. 5, comprising FIG. 5A through FIG. 5C, depicts the
results of blinded and randomized clinical detection of EGFR
mutations in saliva via the system of the present invention. EFIRM
was carried out in duplicates. The absolute values of signal
associated with (FIG. 5A) Exon19 deletion and (FIG. 5B) L858R point
mutation according to patient subgroup are presented
(***p<0.0001, one-way ANOVA and bonferroni post hoc test). (FIG.
5C) Receiver operating characteristic curves for detecting (left to
right) the Exon 19 deletion (AUC=0.94, 95% CI, 0.82 to 1 and the
L858R mutation, respectively (AUC=0.96, 95% CI, 0.90-1).
[0030] FIG. 6, comprising FIG. 6A and FIG. 6B, depicts the results
of experiments demonstrating the correlation of EGFR mutation
statuses between plasma and saliva using system of the present
invention. The scattergram shows the correlation and linear
regression between amperometric currents recorded using plasma and
saliva. Each dot represents data for one patient in (FIG. 6A) the
testing group and (FIG. 6B) the blinded group.
[0031] FIG. 7, comprising FIG. 7A and FIG. 7B, depicts the results
of experiments demonstrating the detection of EGFR mutations from
the plasma of 33 patients with NSCLC by using the system of the
present invention. The amperometric currents of exon 19 deletion
group that were detected using EFIRM with an exon 19 probe were
significantly higher than those in the wild-type and p.L858R mutant
groups (FIG. 7A: 131.3.6.+-.55.76 in p.E746-A750del group versus
9.76.+-.1.77 in wild-type group and 10.26.+-.2.87 in the p.L858R
mutant group, p<0.0001). Similar results were obtained using the
probe for p.L858R (FIG. 7B: 110.6.+-.57.99 in p.L858R mutant group
versus 9.51.+-.2.73 in wild-type group and 9.34.+-.1.86 in
p.E746-A750del group, p<0.0001).
DETAILED DESCRIPTION
[0032] It is to be understood that the figures and descriptions of
the present invention have been simplified to illustrate elements
that are relevant for a clear understanding of the present
invention, while eliminating, for the purpose of clarity, many
other elements found in typical biomarker detection systems and
methods. Those of ordinary skill in the art may recognize that
other elements and/or steps are desirable and/or required in
implementing the present invention. However, because such elements
and steps are well known in the art, and because they do not
facilitate a better understanding of the present invention, a
discussion of such elements and steps is not provided herein. The
disclosure herein is directed to all such variations and
modifications to such elements and methods known to those skilled
in the art.
Definitions
[0033] Unless defined otherwise, all technical and scientific terms
used herein have the same meaning as commonly understood by one of
ordinary skill in the art to which this invention belongs. Although
any methods and materials similar or equivalent to those described
herein can be used in the practice or testing of the present
invention, the preferred methods and materials are described.
[0034] As used herein, each of the following terms has the meaning
associated with it in this section.
[0035] The articles "a" and "an" are used herein to refer to one or
to more than one (i.e., to at least one) of the grammatical object
of the article. By way of example, "an element" means one element
or more than one element.
[0036] "About" as used herein when referring to a measurable value
such as an amount, a temporal duration, and the like, is meant to
encompass variations of .+-.20%, .+-.10%, .+-.5%, .+-.1%, and
.+-.0.1% from the specified value, as such variations are
appropriate. The term "abnormal" when used in the context of
organisms, tissues, cells or components thereof, refers to those
organisms, tissues, cells or components thereof that differ in at
least one observable or detectable characteristic (e.g., age,
treatment, time of day, etc.) from those organisms, tissues, cells
or components thereof that display the "normal" (expected)
respective characteristic. Characteristics which are normal or
expected for one cell or tissue type, might be abnormal for a
different cell or tissue type.
[0037] As used herein the terms "alteration," "defect,"
"variation," or "mutation," refers to a mutation in a gene in a
cell that affects the function, activity, expression (transcription
or translation) or conformation of the polypeptide that it encodes.
Mutations encompassed by the present invention can be any mutation
of a gene in a cell that results in the enhancement or disruption
of the function, activity, expression or conformation of the
encoded polypeptide, including the complete absence of expression
of the encoded protein and can include, for example, missense and
nonsense mutations, insertions, deletions, frameshifts and
premature terminations. Without being so limited, mutations
encompassed by the present invention may alter splicing the mRNA
(splice site mutation) or cause a shift in the reading frame
(frameshift).
[0038] The term "amplification" refers to the operation by which
the number of copies of a target nucleotide sequence present in a
sample is multiplied.
[0039] The term "antibody," as used herein, refers to an
immunoglobulin molecule which specifically binds with an antigen.
Antibodies can be intact immunoglobulins derived from natural
sources or from recombinant sources and can be immunoreactive
portions of intact immunoglobulins. Antibodies are typically
tetramers of immunoglobulin molecules. The antibodies in the
present invention may exist in a variety of forms including, for
example, polyclonal antibodies, monoclonal antibodies, Fv, Fab and
F(ab).sub.2, as well as single chain antibodies and humanized
antibodies (Harlow et al., 1999, In: Using Antibodies: A Laboratory
Manual, Cold Spring Harbor Laboratory Press, NY; Harlow et al.,
1989, In: Antibodies: A Laboratory Manual, Cold Spring Harbor,
N.Y.; Houston et al., 1988, Proc. Natl. Acad. Sci. USA
85:5879-5883; Bird et al., 1988, Science 242:423-426).
[0040] An "antibody heavy chain," as used herein, refers to the
larger of the two types of polypeptide chains present in all
antibody molecules in their naturally occurring conformations.
[0041] An "antibody light chain," as used herein, refers to the
smaller of the two types of polypeptide chains present in all
antibody molecules in their naturally occurring conformations.
.kappa. and .lamda. light chains refer to the two major antibody
light chain isotypes.
[0042] By the term "synthetic antibody" as used herein, is meant an
antibody which is generated using recombinant DNA technology, such
as, for example, an antibody expressed by a bacteriophage as
described herein. The term should also be construed to mean an
antibody which has been generated by the synthesis of a DNA
molecule encoding the antibody and which DNA molecule expresses an
antibody protein, or an amino acid sequence specifying the
antibody, wherein the DNA or amino acid sequence has been obtained
using synthetic DNA or amino acid sequence technology which is
available and well known in the art.
[0043] By the term "specifically binds," as used herein with
respect to an antibody, is meant an antibody which recognizes a
specific antigen, but does not substantially recognize or bind
other molecules in a sample. For example, an antibody that
specifically binds to an antigen from one species may also bind to
that antigen from one or more species. But, such cross-species
reactivity does not itself alter the classification of an antibody
as specific. In another example, an antibody that specifically
binds to an antigen may also bind to different allelic forms of the
antigen. However, such cross reactivity does not itself alter the
classification of an antibody as specific. In some instances, the
terms "specific binding" or "specifically binding," can be used in
reference to the interaction of an antibody, a protein, or a
peptide with a second chemical species, to mean that the
interaction is dependent upon the presence of a particular
structure (e.g., an antigenic determinant or epitope) on the
chemical species; for example, an antibody recognizes and binds to
a specific protein structure rather than to proteins generally. If
an antibody is specific for epitope "A", the presence of a molecule
containing epitope A (or free, unlabeled A), in a reaction
containing labeled "A" and the antibody, will reduce the amount of
labeled A bound to the antibody.
[0044] As used herein, the term "marker" or "biomarker" is meant to
include a parameter which is useful according to this invention for
determining the presence and/or severity of lung cancer.
[0045] The level of a marker or biomarker "significantly" differs
from the level of the marker or biomarker in a reference sample if
the level of the marker in a sample from the patient differs from
the level in a sample from the reference subject by an amount
greater than the standard error of the assay employed to assess the
marker, and preferably at least 10%, and more preferably 25%, 50%,
75%, or 100%.
[0046] The term "control or reference standard" describes a
material comprising none, or a normal, low, or high level of one of
more of the marker (or biomarker) expression products of one or
more the markers (or biomarkers) of the invention, such that the
control or reference standard may serve as a comparator against
which a sample can be compared.
[0047] By the phrase "determining the level of marker (or
biomarker) expression" is meant an assessment of the degree of
expression of a marker in a sample at the nucleic acid or protein
level, using technology available to the skilled artisan to detect
a sufficient portion of any marker expression product.
[0048] "Differentially increased expression" or "up regulation"
refers to biomarker product levels which are at least 10% or more,
for example, 20%, 30%, 40%, or 50%, 60%, 70%, 80%, 90% higher or
more, and/or 1.1 fold, 1.2 fold, 1.4 fold, 1.6 fold, 1.8 fold, 2.0
fold higher or more, and any and all whole or partial increments
therebetween than a control.
[0049] "Differentially decreased expression" or "down regulation"
refers to biomarker product levels which are at least 10% or more,
for example, 20%, 30%, 40%, or 50%, 60%, 70%, 80%, 90% lower or
less, and/or 2.0 fold, 1.8 fold, 1.6 fold, 1.4 fold, 1.2 fold, 1.1
fold or less lower, and any and all whole or partial increments
therebetween than a control.
[0050] A "disease" is a state of health of an animal wherein the
animal cannot maintain homeostasis, and wherein if the disease is
not ameliorated then the animal's health continues to
deteriorate.
[0051] As used herein, an "instructional material" includes a
publication, a recording, a diagram, or any other medium of
expression which can be used to communicate the usefulness of a
component of the invention in a kit for detecting biomarkers
disclosed herein. The instructional material of the kit of the
invention can, for example, be affixed to a container which
contains the component of the invention or be shipped together with
a container which contains the component. Alternatively, the
instructional material can be shipped separately from the container
with the intention that the instructional material and the
component be used cooperatively by the recipient.
[0052] The term "label" when used herein refers to a detectable
compound or composition that is conjugated directly or indirectly
to a probe to generate a "labeled" probe. The label may be
detectable by itself (e.g. radioisotope labels or fluorescent
labels) or, in the case of an enzymatic label, may catalyze
chemical alteration of a substrate compound or composition that is
detectable (e.g., avidin-biotin). In some instances, primers can be
labeled to detect a PCR product.
[0053] The "level" of one or more biomarkers means the absolute or
relative amount or concentration of the biomarker in the
sample.
[0054] The term "marker (or biomarker) expression" as used herein,
encompasses the transcription, translation, post-translation
modification, and phenotypic manifestation of a gene, including all
aspects of the transformation of information encoded in a gene into
RNA or protein. By way of non-limiting example, marker expression
includes transcription into messenger RNA (mRNA) and translation
into protein, as well as transcription into types of RNA such as
transfer RNA (tRNA) and ribosomal RNA (rRNA) that are not
translated into protein.
[0055] "Measuring" or "measurement," or alternatively "detecting"
or "detection," means assessing the presence, absence, quantity or
amount (which can be an effective amount) of either a given
substance within a clinical or subject-derived sample, including
the derivation of qualitative or quantitative concentration levels
of such substances, or otherwise evaluating the values or
categorization of a subject's clinical parameters.
[0056] The terms "patient," "subject," "individual," and the like
are used interchangeably herein, and refer to any animal, or cells
thereof whether in vitro or in situ, amenable to the methods
described herein. In certain non-limiting embodiments, the patient,
subject or individual is a human.
[0057] As used herein, the term "providing a prognosis" refers to
providing a prediction of the probable course and outcome of lung
cancer, including prediction of severity, duration, chances of
recovery, etc. The methods can also be used to devise a suitable
therapeutic plan, e.g., by indicating whether or not the condition
is still at an early stage or if the condition has advanced to a
stage where aggressive therapy would be ineffective.
[0058] A "reference level" of a biomarker means a level of the
biomarker that is indicative of a particular disease state,
phenotype, or lack thereof, as well as combinations of disease
states, phenotypes, or lack thereof. A "positive" reference level
of a biomarker means a level that is indicative of a particular
disease state or phenotype. A "negative" reference level of a
biomarker means a level that is indicative of a lack of a
particular disease state or phenotype.
[0059] "Sample" or "biological sample" as used herein means a
biological material isolated from an individual. The biological
sample may contain any biological material suitable for detecting
the desired biomarkers, and may comprise cellular and/or
non-cellular material obtained from the individual.
[0060] "Standard control value" as used herein refers to a
predetermined amount of a particular protein or nucleic acid that
is detectable in a sample, such as a saliva sample, either in whole
saliva or in saliva supernatant. The standard control value is
suitable for the use of a method of the present invention, in order
for comparing the amount of a protein or nucleic acid of interest
that is present in a saliva sample. An established sample serving
as a standard control provides an average amount of the protein or
nucleic acid of interest in the saliva that is typical for an
average, healthy person of reasonably matched background, e.g.,
gender, age, ethnicity, and medical history. A standard control
value may vary depending on the protein or nucleic acid of interest
and the nature of the sample (e.g., whole saliva or
supernatant).
[0061] Throughout this disclosure, various aspects of the invention
can be 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, 2.7, 3, 4, 5, 5.3, 6 and any whole and partial
increments therebetween. This applies regardless of the breadth of
the range.
Description
[0062] The present invention relates to a rapid and accurate
polymer-based electrochemical platform array for single or
multi-biomarker detection from a biological sample, such as a
saliva sample or blood sample, that are indicative of lung cancer.
While the present invention is described generally for the testing
of a saliva sample or blood sample, it should be appreciated that
any biological fluid sample may be used, or even other tissue
types, provided such alternative sample types carry the targeted
markers to be analyzed. Non-limiting examples of such markers
include all saliva-based lung cancer gene mutations, such as one or
more mutations in epidermal growth factor receptor (EGFR), or any
other markers associated with or indicative of lung cancer. For
example, non-limiting examples of such detectable nucleic acids, or
detectable mutations in genes include those associated with KRAS,
BRAF, CCNI, FGF19, FRS2, GREB1 and LZTS1. It should be appreciated
that any number of biomarkers can be integrated to the assay
platform, including, without limitation, 1, 2, 4, 8, 16, 32 or 64
biomarkers per array.
[0063] The noninvasive detection of lung cancer in a subject via
the present invention enables clinicians to identify the presence
of lung cancer in a fast, economical and non-invasive manner. As
contemplated herein, the present invention includes a multiplexing
electrochemical sensor for detecting lung cancer biomarkers. The
device utilizes a small sample volume with high accuracy. In
addition, multiple markers can be measured simultaneously on the
device with single sample loading. The device may significantly
reduce the cost to the health care system, by decreasing the burden
of patients returning to clinics and laboratories.
[0064] In one embodiment, the electrochemical sensor is an array of
electrode chips (GeneFluidics, USA). As shown in FIG. 1, each unit
of the array has a working electrode, a counter electrode, and a
reference electrode. The three electrodes may be constructed of
bare gold or other conductive material before the reaction, such
that the specimens may be immobilized on the working electrode.
Electrochemical current can be measured between the working
electrode and counter electrode under the potential between the
working electrode and the reference electrode. The potential
profile can be a constant value, a linear sweep, or a cyclic square
wave, for example. An array of plastic wells may be used to
separate each three-electrode set, which helps avoid the cross
contamination between different sensors. A conducting polymer may
also be deposited on the working electrodes as a supporting film,
and in some embodiments, as a surface to functionalize the working
electrode. As contemplated herein, any conductive polymer may be
used, such as polypyrroles, polyanilines, polyacetylenes,
polyphenylenevinylenes, polythiophenes and the like.
[0065] In a preferred embodiment, a cyclic square wave electric
field is generated across the electrode within the sample well. In
certain embodiments, the square wave electric field is generated to
aid in polymerization of one or more capture probes to the polymer
of the sensor. In certain embodiments, the square wave electric
field is generated to aid in the hybridization of the capture
probes with the marker and/or detector probe. The positive
potential in the csw E-field helps the molecules accumulate onto
the working electrode, while the negative potential removes the
weak nonspecific binding, to generate enhanced specificity.
Further, the flapping between positive and negative potential
across the cyclic square wave also provides superior mixing during
incubation, without disruption of the desired specific binding,
which accelerates the binding process and results in a faster test
or assay time. In one embodiment, a square wave cycle may consist
of a longer low voltage period and a shorter high voltage period,
to enhance binding partner hybridization within the sample. While
there is no limitation to the actual time periods selected,
examples include 0.15 to 60 second low voltage periods and 0.1 to
60 second high voltage periods. In a preferred embodiment, each
square-wave cycle consists of 1 s at low voltage and 1 s at high
voltage. For hybridization, the low voltage may be around -200 mV
and the high voltage may be around +500 mV. In some embodiments,
the total number of square wave cycles may be between 2-50. In a
preferred embodiment, 5 cyclic square-waves are applied for each
surface reaction. With the csw E-field, both the polymerization and
hybridization are finished on the same chip within minutes. In some
embodiments, the total detection time from sample loading is less
than 30 minutes. In other embodiments, the total detection time
from sample loading is less than 20 minutes. In other embodiments,
the total detection time from sample loading is less than 10
minutes. In other embodiments, the total detection time from sample
loading is less than 5 minutes. In other embodiments, the total
detection time from sample loading is less than 2 minutes. In other
embodiments, the total detection time from sample loading is less
than 1 minute.
[0066] A multi-channel electrochemical reader (GeneFluidics)
controls the electrical field applied onto the array sensors and
reports the amperometric current simultaneously. In practice,
solutions can be loaded onto the entire area of the three-electrode
region including the working, counter, and reference electrodes,
which are confined and separated by the array of plastic wells.
After each step, the electrochemical sensors can be rinsed with
ultrapure water or other washing solution and then dried, such as
under pure N.sub.2. In some embodiments, the sensors are single
use, disposable sensors. In other embodiment, the sensors are
reusable.
[0067] In one embodiment, the present invention is based on the
affinity between a capture probe and a detector probe, as shown in
FIG. 1. As contemplated herein, the assay platform may be organized
as any type of affinity binding assay or immunoassay as would be
understood by those skilled in the art. In another embodiment, the
present invention includes a single platform for multiple lung
cancer biomarker measurements, instead of a single marker.
Currently, there is no such technology or device available for this
purpose. In another embodiment, the present invention creates
tremendous efficiencies in that it is simple, rapid and robust. For
example, only small sample volumes are needed (e.g., 10 .mu.l) and
less than 10 minutes run time are needed. Multiple marker levels
may be provided by the device. By providing statistical analysis
the user may have an estimate of their risk, and by utilizing
available networking systems, the results can be quickly
transmitted for review by a clinician for further assessment.
[0068] For example, paired probes (capture and detector) specific
for a mutation can be designed, such as for a deletion mutation, or
for a point mutation. In the example of FIG. 1 and the experimental
presented herein, two sets of probes, one for a deletion mutation
and one for a point mutation, are used. The detector probes can be
labeled, such as with fluorescein isothiocyanate or any other label
known in the art. The capture probe is first copolymerized onto the
bare gold electrode by applying a cyclic square wave electric
field. For example, for each cycle during copolymerization, the
electric field can be set to +350 mV for 1 s and +950 mV for 1 s.
In total, polymerization may proceed for 5 cycles of 10 s, or
however long is deemed necessary.
[0069] After polymerization, the sensor chip can be rinsed and
dried for subsequent sample measurement. Samples, such as a
cell-culture medium, a blood sample or a saliva sample, can be
mixed with the detector probes and transferred onto the electrodes.
Hybridization is then carried out at low and high voltage cycles,
such as -200 mV for 1 s and +500 mV for 1 s. The total
hybridization time can be 5 cycles for 10 s, for example. Next, the
label is detected based on the label type. For example, an
anti-fluorescein antibody conjugated to horseradish peroxidase in
casein-phosphate-buffered saline can be used, and the
3,3',5,5'-tetramethylbenzidine substrate for horseradish peroxidase
can be loaded, and the amperometric signal measured.
[0070] Capture probes embedded in the conductive polymer or
otherwise used to functionalize the working electrode surface, and
detector probes mixed with the sample may be constructed according
to any protocol known in the art for the generation of probes.
[0071] The capture probe or detector probe of the sensor may be any
one of a nucleic acid, protein, small molecule, and the like, which
specifically binds one or more of the markers of interest. For
example, in a particular embodiment, the capture probe and detector
probe are oligonucleotides or polynucleotides comprising a region
that is substantially complementary to one or more nucleic acid
markers of the invention. In one embodiment, the capture probe and
detector probe comprise a region that is substantially
complementary to each other. That is, in one embodiment, the
capture probe comprises a region that is substantially
complementary to a region of the detector probe. Methods for
designing and formulating oligonucleotide probes are well-known in
the art.
[0072] In one embodiment, the marker comprises a variable region,
where the variable region may comprise a mutation, such as a
deletion, substitution, point mutation, and the like, associated
with the disease. In one embodiment, the capture probe is designed
to hybridize to a conserved region of the nucleic acid marker (i.e.
present in wild-type and mutant isoforms). In one embodiment, the
detector probe is designed to hybridize to the nucleic acid
sequence of the variable region having a disease-associated
mutation of the marker, thereby detecting the mutation in the
sample. In another embodiment, the detector probe is designed to
hybridize to the nucleic acid sequence of the variable region
having a wild-type or non-mutated sequence, thereby detecting the
presence of the wildtype marker in the sample. In one embodiment,
the capture probe is designed to hybridize to the wildtype or
mutated variable region, while the detector probe is designed to
hybridize to the conserved region.
[0073] For example, in one embodiment, the capture probe comprises
a nucleotide sequence that is substantially complementary to a
conserved region of a nucleic acid encoding EGFR. In one
embodiment, the detector probe comprises a nucleotide sequence that
is substantially complementary to a variable region of a nucleic
acid encoding EGFR, where the variable region encodes the wild-type
amino acid sequence of EGFR. In one embodiment, the detector probe
comprises a nucleotide sequence that is substantially complementary
to a variable region of a nucleic acid encoding EGFR, where the
variable region encodes the disease-associated mutant sequence of
EGFR. For example, in certain embodiments, the detector probe
comprises a nucleotide sequence that is substantially complementary
to a variable region of a nucleic acid encoding EGFR, where the
variable region encodes the disease-associated deletion mutant or
point mutation of EGFR.
[0074] In one embodiment, when the present invention is used for
detecting a nucleic acid encoding the E746-A750 deletion mutant of
EGFR, the capture probe comprises a nucleic acid molecule
comprising the nucleotide sequence of SEQ ID NO: 1. In one
embodiment, when the present invention is used for detecting a
nucleic acid encoding the L858R point mutation of EGFR, the capture
probe comprises a nucleic acid molecule comprising the nucleotide
sequence of SEQ ID NO: 3.
[0075] In one embodiment, when the present invention is used for
detecting a nucleic acid encoding the E746-A750 deletion mutant of
EGFR, the detector probe comprises a nucleic acid molecule
comprising the nucleotide sequence of SEQ ID NO: 2. In one
embodiment, when the present invention is used for detecting a
nucleic acid encoding the L858R point mutation of EGFR, the
detector probe comprises a nucleic acid molecule comprising the
nucleotide sequence of SEQ ID NO: 4.
[0076] In one embodiment, the detector probe comprises a detectable
label which induces a change in current of the sensor, thereby
indicating the hybridization of the detector probe, and associated
marker, with the capture probe. In certain embodiments, the
detectable label itself may be sufficient to alter the current of
the sensor. In certain embodiments, the detectable label induces
the change in current when it comes into contact with an exogenous
reactant. For example, the detectable label may react with the
reactant to produce a local change sensed by the electrodes of the
sensor to produce an amperometric signal. Therefore, in certain
embodiments, the reactant is added to the sensor prior, during, or
after the application of the sample to the sensor.
[0077] In certain embodiments, the detectable label is directly
conjugated to the detector probe. In another embodiment, the
detectable label is bound to the detector probe via an intermediate
tag or label of the probe. For example, in one embodiment, the
detector probe comprises a tag, label, or epitope, which can be
used to bind to an antibody or other binding compound harboring the
detectable label described above.
[0078] Examples of detectable labels and reactants to produce a
local change in an electrochemical sensor are well known in the
art. In one embodiment, the detectable label comprises HRP and the
reactant is TMB, which react to generate an amperometric signal. In
another embodiment, the detectable label comprises urease, while
the reactant comprises urea.
[0079] There is no limitation to the concentrations of such probes
used, and may be optimized as needed by the user.
[0080] Due to the enhanced sensitivity of the present invention,
very small volumes may be used to perform the desired assays. For
example, the biological sample size from the subject may be between
5-100 microliters. In a preferred embodiment, the sample size need
only be about 40 microliters. There is no limitation to the actual
or final sample size to be tested.
[0081] The present invention also relates to a method of detecting
one or more markers associated with or indicative of lung cancer in
a subject. In one embodiment, the method may be performed as a
hybridization assay and includes the steps of obtaining a sample
from the subject, adding an detector probe labeled with a
detectable moiety directed against a targeted marker of lung cancer
to the sample, applying the sample to an electrode chip coated with
a conducting polymer previously embedded or functionalized with the
capture probe, and measuring the current in the electrode chip. The
detectable moiety may be measured, or the magnitude of the current
in the sample may be measured, to determine the presence or absence
of the marker in the sample. In certain embodiments, hybridization
of the marker to the electrode of the sensor results in an increase
in current or negative current. For example, in one embodiment,
hybridization results in a current in the range of about -10 nA to
about -1000 nA.
[0082] The present invention provides a method for diagnosing lung
cancer in a subject. Accordingly, the present invention features
methods for identifying subjects who are at risk of developing lung
cancer, including those subjects who are asymptomatic or only
exhibit non-specific indicators of lung cancer by detection of the
biomarkers disclosed herein. These biomarkers are also useful for
monitoring subjects undergoing treatments and therapies for lung
cancer, and for selecting or modifying therapies and treatments
that would be efficacious in subjects having lung cancer, wherein
selection and use of such treatments and therapies slow the
progression of lung cancer, or prevent their onset.
[0083] In certain embodiments, the biomarkers detected by way of
the system and method of the invention include, but are not limited
to nucleic acids, or detectable mutations in genes including those
associated with EGFR, KRAS, BRAF, CCNI, FGF19, FRS2, GREB1 and
LZTS1. In certain embodiments, the nucleic acid biomarkers comprise
one or more disease-associated mutations, including, insertions,
deletions, substitutions, translocations, point mutations, single
nucleotide variants (SNVs), and the like. The present invention may
be used to detect any disease-associated biomarker or
disease-associated mutation known in the art or discovered in the
future. Exemplary mutations present in various forms of cancer may
be found, for example, in the Catalogue of Somatic Mutations in
Cancer (COSMIC) (Wellcome Trust Sanger Institute).
[0084] In certain embodiments, the biomarkers detected by way of
the system and method of the present invention comprise mutant EGFR
and nucleic acid molecules encoding mutant EGFR. For example,
various EGFR mutants are associated with lung cancer, including but
not limited deletions in exon 19, point mutations in exons 18-21,
and in-frame insertions or duplications that occur mostly in exon
20.
[0085] Exemplary deletions in exon 19 include for example deletion
of one or more amino acids in the region of K745-S752, including
but not limited to, E746-A750 deletion, K745-E749 deletion,
E746-E749 deletion, and E746-R748 deletion. Exemplary point
mutations of EGFR include, but are not limited to G719X, L858R,
T790M, and L861Q.
[0086] The present invention may be used to detect any such
mutation, as the capture probe and/or detector probe may be
specifically designed to hybridize to a nucleic acid molecule
encoding the mutant protein of interest.
[0087] The invention provides improved diagnosis and prognosis of
lung cancer. The risk of developing lung cancer can be assessed by
measuring one or more of the biomarkers described herein, and
comparing the measured values to reference or index values. Such a
comparison can be undertaken with mathematical algorithms or
formula in order to combine information from results of multiple
individual biomarkers and other parameters into a single
measurement or index. Subjects identified as having an increased
risk of lung cancer can optionally be selected to receive treatment
regimens, such as administration of prophylactic or therapeutic
compounds or treatments to prevent, treat or delay the onset of
lung cancer.
[0088] Identifying a subject before they develop lung cancer
enables the selection and initiation of various therapeutic
interventions or treatment regimens in order to delay, reduce or
prevent the development or severity of the cancer. In certain
instances, monitoring the levels of at least one biomarker also
allows for the course of treatment of lung cancer to be monitored.
For example, a sample can be provided from a subject undergoing
treatment regimens or therapeutic interventions, e.g., drug
treatments, radiation, chemotherapy, etc. for lung cancer. Samples
can be obtained from the subject at various time points before,
during, or after treatment.
[0089] The biomarkers of the present invention can thus be used to
generate a biomarker profile or signature of subjects: (i) who do
not have and are not expected to develop lung cancer and/or (ii)
who have or expected to develop lung cancer. The biomarker profile
of a subject can be compared to a predetermined or reference
biomarker profile to diagnose or identify subjects at risk for
developing lung cancer, to monitor the progression of disease, as
well as the rate of progression of disease, and to monitor the
effectiveness of lung cancer treatments. Data concerning the
biomarkers of the present invention can also be combined or
correlated with other data or test results for lung cancer,
including but not limited to imaging data, medical history and any
relevant family history.
[0090] The present invention also provides methods for identifying
agents for treating lung cancer that are appropriate or otherwise
customized for a specific subject. In this regard, a test sample
from a subject, exposed to a therapeutic agent, drug, or other
treatment regimen, can be taken and the level of one or more
biomarkers can be determined. The level of one or more biomarkers
can be compared to a sample derived from the subject before and
after treatment, or can be compared to samples derived from one or
more subjects who have shown improvements in risk factors as a
result of such treatment or exposure.
[0091] In one embodiment, the invention is a method of diagnosing
lung cancer. In one embodiment, the method includes determining the
stage or severity of lung cancer. In some embodiments, these
methods may utilize a biological sample (such as urine, saliva,
blood, serum, plasma, amniotic fluid, or tears), for the detection
of one or more markers of the invention in the sample. Frequently
the sample will be a "clinical sample" which is a sample derived
from a patient. In one embodiment, the biological sample is a blood
sample. In certain embodiments, the biological sample is a serum
sample or a plasma sample, derived from a blood sample of the
subject.
[0092] In one embodiment, the method comprises detecting one or
more markers in a biological sample of the subject. Preferably, the
biological sample is saliva. In various embodiments, the level of
one or more of markers of the invention in the biological sample of
the subject is compared with the level of a corresponding biomarker
in a comparator. Non-limiting examples of comparators include, but
are not limited to, a negative control, a positive control, an
expected normal background value of the subject, a historical
normal background value of the subject, an expected normal
background value of a population that the subject is a member of,
or a historical normal background value of a population that the
subject is a member of.
[0093] In another embodiment, the invention is a method of
monitoring the progression of lung cancer in a subject by assessing
the level of one or more of the markers of the invention in a
biological sample of the subject.
[0094] In various embodiments, the subject is a human subject, and
may be of any race, sex and age.
[0095] Information obtained from the methods of the invention
described herein can be used alone, or in combination with other
information (e.g., disease status, disease history, vital signs,
blood chemistry, etc.) from the subject or from the biological
sample obtained from the subject.
[0096] In other various embodiments of the methods of the
invention, the level of one or more markers of the invention is
determined to be increased when the level of one or more of the
markers of the invention is increased by at least 10%, by at least
20%, by at least 30%, by at least 40%, by at least 50%, by at least
60%, by at least 70%, by at least 80%, by at least 90%, or by at
least 100%, when compared to with a comparator control.
[0097] In the methods of the invention, a biological sample from a
subject is assessed for the level of one or more of the markers of
the invention in the biological sample obtained from the patient.
The level of one or more of the markers of the invention in the
biological sample can be determined by assessing the amount of
polypeptide of one or more of the biomarkers of the invention in
the biological sample, the amount of mRNA of one or more of the
biomarkers of the invention in the biological sample, the amount of
DNA of one or biomarkers of the invention in the biological sample,
the amount of enzymatic activity of one or more of the biomarkers
of the invention in the biological sample, or a combination
thereof.
[0098] The present invention further includes an assay kit
containing the electrochemical sensor array and instructions for
the set-up, performance, monitoring, and interpretation of the
assays of the present invention. Optionally, the kit may include
reagents for the detection of at least one of the biomarkers. The
kit may also optionally include the sensor reader.
EXPERIMENTAL EXAMPLES
[0099] The invention is further described in detail by reference to
the following experimental examples. These examples are provided
for purposes of illustration only, and are not intended to be
limiting unless so specified. Thus, the invention should in no way
be construed as being limited to the following examples, but
rather, should be construed to encompass any and all variations
which become evident as a result of the teaching provided
herein.
[0100] Without further description, it is believed that one of
ordinary skill in the art can, using the preceding description and
the following illustrative examples, make and utilize the present
invention and practice the claimed methods. The following working
examples therefore, specifically point out exemplary embodiments of
the present invention, and are not to be construed as limiting in
any way the remainder of the disclosure.
Example 1 Noninvasive EGFR Gene Mutation Detection in Patients with
Lung Cancer
[0101] The detection of EGFR mutations by the system of the present
invention was developed and validated in four consecutive phases.
First, in vitro spike-in experiments were carried out with human
lung-cancer cell lines with two specific human EGFR mutations:
c.2236_2250del15, which encodes the p.E746-A750 mutation (the major
form of the exon 19 deletion) that is present in the HCC827 cell
line, and c.2573T>G, which encodes the p.L858R mutation in the
NCI-H1975 cancer cell line. Second, mutant EGFR DNA was detected in
body fluids as a function of lung tumor burden in a mouse lung
cancer xenograph model. Plasma from mice xenografted with the human
lung cancer cell line NCI-H1975 (carrying the EGFR L858R mutation)
was tested at various stages of tumor progression. Third, the
system was used for the detection of EGFR mutations in saliva and
plasma of NSCLC patients. Lastly, EGFR mutation detection was
validated in saliva from human lung cancer patients in a blinded
clinical study.
[0102] The materials and methods employed in these experiments are
now described.
Cell Culture and DNA Isolation
[0103] Human lung cancer cell lines NCI-H1975 and HCC827 were
obtained from the American Type Culture Collection (Manassas, Va.,
USA). Cells were propagated in RPMI-1640 medium with 10% fetal
bovine serum. Genomic DNA was isolated with the DNeasy Blood &
Tissue Kit (Qiagen, Valencia, Calif., USA), and DNA concentration
was determined using a Nanodrop.TM. 8000 spectrophotometer (Thermo
Scientific, Wilmington, Del., USA).
Mouse Lung Cancer Xenograft Model
[0104] Female nu/nu mice .about.6-8 weeks old were obtained from
Charles River Labs (MA, USA). Animals were housed under standard
clean-room conditions in accordance with the guidelines of the
Pfizer Institutional Animal Care and Use Committee. NCI-H1975 cells
(2.times.10.sup.6 cells) were re-suspended in serum-free Matrigel
at a 1:1 ratio and immediately implanted subcutaneously in the
right flank of each animal (n=9).
Patient Samples
[0105] To be eligible for inclusion in this study, patients were
required to have pathology-confirmed stage III or IV lung
adenocarcinoma. Patients treated at NCKUH (Tainan, Taiwan) from
June 2012 to December 2013 were enrolled. This study was approved
by the Research Ethics Committee of NCKUH. All patients provided
informed consent to participate in this study and gave permission
for the use of their tumor tissues and the collection of their
saliva for EGFR mutation analysis.
[0106] To ensure blinding of the analysis during the validation
phase, the center in charge of distribution (MD Anderson Cancer
Center (Houston, Tex.)) assigned random numbers to saliva samples
from NCKUH and distributed the codes to UCLA for testing. The
person who assigned the random numbers at the distribution center
was not the same person who performed the testing at UCLA, nor the
person who collected the saliva at NCKUH, thus ensuring complete
blinding of the samples. The code on the saliva was broken only
after the UCLA lab submitted the data.
Collection of Mouse Plasma
[0107] To isolate plasma from mice, blood was collected when tumors
were 100-300 mm.sup.3, 500-900 mm.sup.3, or >1000 mm.sup.3 at
11, 16, and 19 days post-transplantation. Three mice were
sacrificed per group, including naive control mice. Animals were
placed under anesthesia (isoflurane-oxygen mixture) and blood was
acquired via cardiac puncture. Approximately 1 mL of blood was
collected in EDTA-containing Vacutainer tubes (BD Biosciences, San
Jose, USA) and spun at 1900.times.g for 10 min at 4.degree. C. The
plasma supernatant was aspirated, transferred to a 1.5-mL
microcentrifuge tube, and centrifuged at 16,000.times.g for 10 min
at 4.degree. C. to remove additional cellular debris. The plasma
supernatant was aspirated and transferred to another
microcentrifuge tube and stored at -80.degree. C. until use.
PCR-Based Analysis of EGFR Mutations
[0108] Tumor tissue from lung tumors, metastatic sites, and
malignant effusion cell blocks were obtained for EGFR mutation
analysis. Tissue samples that consisted of >80% tumor content as
determined via microscopy with hematoxylin and eosin staining were
selected for the study. Pleural effusion fluid was collected and
centrifuged at 250.times.g for 10 min at 4.degree. C., and the cell
pellet was frozen. Sample processing, from sampling to freezing,
required <2 h (39). Genomic DNA was extracted from cell lysates
or tumor paraffin blocks using the QIAamp DNA Mini Kit (Qiagen) and
used in EGFR mutation analysis, as described previously (40).
Saliva Collection
[0109] Since the exon 19 deletion and the exon 21 L858R point
mutation represent 90% of EGFR sensitizing mutations (Sharma S V,
Bell D W, Settleman J, Haber D A. Epidermal growth factor receptor
mutations in lung cancer. Nature reviews Cancer 2007; 7: 169-181;
Sequist L V, Bell D W, Lynch T J, Haber D A. Molecular predictors
of response to epidermal growth factor receptor antagonists in
non-small-cell lung cancer. Journal of clinical oncology: official
journal of the American Society of Clinical Oncology 2007; 25:
587-595), only patients confirmed to be wildtype or harboring these
mutations were allowed to enroll in the study. Unstimulated whole
saliva was collected and processed according to previously
established protocols (Li Y, St John M A, Zhou X, et al. Salivary
transcriptome diagnostics for oral cancer detection. Clin Cancer
Res 2004; 10: 8442-8450). Briefly, saliva samples were kept on ice
during collection and were then centrifuged at 2,600.times.g for 15
min at 4.degree. C. The supernatant was removed from the pellet,
treated with RNase inhibitor (Superase-In, Ambion Inc., Austin,
Tex., USA), and stored at -80.degree. C. prior to use.
Detection of EGFR Mutations in Body Fluids Via System Platform
[0110] FIG. 1 illustrates the core technology of the system of the
present invention. The sensor is a conducting polymer-based
electrochemical chip with an array of 16 bare-gold electrode chips
(GeneFluidics, Los Angeles, USA). Each unit of the array has a
working electrode, a counter electrode, and a reference electrode.
The 16-channel electrochemical (EC) reader (GeneFluidics) controls
the electrical field applied onto the 16 array sensors and
simultaneously reports the amperometric current.
[0111] Paired probes (capture and detector; Sigma, St. Louis, USA)
specific for the two TKI-sensitive mutations were designed for the
platform as follows: capture probe for the exon 19 deletion, 5'-TGT
TGC TTC CTT GAT AGC GAC G-3' (SEQ ID NO: 1); detector probe for the
exon 19 deletion, 5'-GGA ATT TTA ACT TTC TCA CCT-3'(SEQ ID NO: 2);
capture probe for the L858R point mutation: 5'-CAG TTT GGC CCG CCC
AAA ATC-3'(SEQ ID NO: 3); detector probe for the L858R mutation:
5'-TTG ACA TGC TGC GGT GTT TTC A-3' (SEQ ID NO: 4). As used in
these experiments, the detector probes were labeled with
fluorescein isothiocyanate (FITC) at their 3' end. Twenty
microliters of the capture probe (100 nM) were first copolymerized
with pyrrole (Sigma) and 100 .mu.L of 3 M KCl (Mettler Toledo,
Columbus, USA) in 1.times. phosphate-buffered saline (pH 7.5;
Invitrogen, Grand Island, USA) onto the bare gold electrode by
applying a cyclic square wave electric field. KCl was added at a
final concentration of 300 mM to achieve high ionic strength. For
each cycle, the electric field was set to +350 mV for 1 s and +950
mV for 1 s. In total, polymerization proceeded for 5 cycles of 10
s.
[0112] After polymerization, the sensor chip was rinsed with
deionized water and dried. Samples, such as cell-culture medium or
40 .mu.L samples of blood or saliva, were then mixed with 5 .mu.L
of the detector probes in 955 .mu.L Tris-HCl buffer (Invitrogen)
and transferred onto the electrodes. Hybridization was carried out
at -200 mV for 1 s and +500 mV for 1 s. The total polymerization
time was again 5 cycles for 10 s. Next, 150 U/mL of
anti-fluorescein antibody conjugated to horseradish peroxidase
(1:1000 dilution; Roche, Indianapolis, USA) in
casein-phosphate-buffered saline (Invitrogen) was added. Finally,
the 3,3',5,5'-tetramethylbenzidine substrate for horseradish
peroxidase was loaded, and the amperometric signal was measured.
The total detection time was <10 min and required only 20-40
.mu.L of biological sample.
Statistical Analysis
[0113] To evaluate the performance of the system of the present
invention in detecting EGFR mutations, the receiver operating
characteristic curve was plotted for each probe and the AUC was
calculated and its 95% confidence interval. All analyses were
performed using SAS 9.3 TS Level 1M1 (DeLong E R, DeLong D M,
Clarke-Pearson D L. Comparing the areas under two or more
correlated receiver operating characteristic curves: a
nonparametric approach. Biometrics 1988; 44: 837-845).
[0114] The results of the experiments are now described
Optimization of EGFR Mutations Detection Using Lung-Cancer Cell
Lines
[0115] The system as depicted in FIG. 1 was optimized to detect the
following EGFR mutations: c.2573T>G in exon 21 (encoding
p.L858R) and c.2236_2250del15 in exon 19 (coding for a 15-base pair
deletion resulting in mutation E746-A750del). Genomic DNA samples
from human lung-cancer cell lines NCI-H1975 and HCC827, harboring
the respective mutations, were used for detection
optimizations.
[0116] The electrical field profile was first optimized for the
appropriate number of hybridization cycles (FIG. 2A and FIG. 2B).
The hybridization signal increased rapidly after two cycles of
electrical waves. After five cycles, the perfect match signal
reached a plateau, while the mismatch sequences generated only
background signal levels. Therefore, the optimized hybridization
cycle was defined as five for all subsequent studies.
[0117] The specificity and sensitivity of the system for detecting
the respective EGFR mutations were investigated by decreasing the
ratio of mutant EGFR DNA to wild-type EGFR DNA (FIG. 2C-FIG. 2F).
For the p.E746-A750del, as little as 0.1% mutant DNA was detected
in the presence of wild-type DNA. For the p.L858R point mutation,
as little as 1% mutant DNA was detected when a control sample was
used. Ten microliters of 2 ng/.mu.L DNA were used for these
experiments. These data demonstrate that system of the present
invention can detect EGFR mutations with high sensitivity and
specificity.
Detection of EGFR Mutation in Plasma of a Mouse Model with
Xenografted Lung Cancer
[0118] Plasma was banked from a mouse model xenografted with the
human lung cancer cell line NCI-H1975 that harbors the EGFR point
mutation p.L858R at increasing tumor burden in 4 different
settings: (1) no tumor (non-xenograft mice), (2) small tumors
(100-500 mm.sup.3), (3) medium tumors (500-900 mm.sup.3), and (4)
large tumors (>900 mm.sup.3). Mice xenografted with the HCT 116
cell line, which carried the EGFR wild type, were used as a control
(Bamford et al., 2004, Br J Cancer, 91: 355-358). To determine if
the optimized system can detect the EGFR mutation in plasma of
these xenografted mice, forty microliters of mouse plasma were
assayed in triplicate with the system at defined time intervals of
day 0, 11, 16 and 19 (FIG. 3A). A positive relationship between
electrochemical current and tumor size was observed (FIG. 3B-FIG.
3D; (b) 10 .mu.l of plasma (R=0.86), (c) 20 .mu.l of plasma
(R=0.98), and (d) 40 .mu.l of plasma (R=0.95)). Increased plasma
volume yielded better discrimination of different tumor sizes (FIG.
3B-FIG. 3D). Naive mice were associated with the lowest amount of
signal (FIG. 3B-FIG. 3D). Small tumors (100-500 mm.sup.3) were
associated with signal levels that significantly differed from the
naive group, and larger tumors had higher current signals (FIG.
3B-FIG. 3D). After 19 days of growth, DNA from mice with large
tumors generated electrochemical current on the order of several
hundred nA (FIG. 3C and FIG. 3D). These results indicate that the
system of the present invention can detect and monitor the
progressive tumor development from the plasma of xenografted mice.
Amperometric readouts are provided in FIG. 3E-FIG. 3G.
Detection of EGFR Mutations in Plasma and Saliva from NSCLC
Patients Using the Platform Assay
[0119] Experiments were conducted to examine whether the two most
common TKI-sensitive EGFR mutations (p.L858R and p.E746-A750del)
can be detected by the system of present invention in plasma and
saliva of NSCLC patients. For each NSCLC subject, tissue, plasma
and saliva were collected at National Cheng Kung University
Hospital (NCKUH). After sample collection, biopsy-based EGFR
genotyping was performed at NCKUH. plasma and saliva-based
detection of the two EGFR mutations were assayed by EFIRM at
University of California, Los Angeles (UCLA).
[0120] Twenty-two NSCLC patients (12 men and 10 women, mean age of
62.0.+-.12.7, mostly non-smokers) met the enrollment criteria and
were enrolled (Table 1). The EGFR mutation rate was comparable to
other study in detecting EGFR mutation in Asia lung adenocarcinoma
ranging from 38% (Huang Y S, Yang J J, Zhang X C, Yang X N, Huang Y
J, Xu C R, Zhou Q, Wang Z, Su J, Wu Y L. Impact of smoking status
and pathologic type on epidermal growth factor receptor mutations
in lung cancer. Chin Med J (Engl) 2011; 124:2457-2460) to 55% (Ho H
L, Chang F P, Ma H H, Liao L R, Chuang Y T, Chang-Chien Y C, Lin K
Y, Chou T Y. Molecular diagnostic algorithm for epidermal growth
factor receptor mutation detection in asian lung adenocarcinomas:
Comprehensive analyses of 445 taiwanese patients with
immunohistochemistry, per-direct sequencing and scorpion/arms
methods. Respirology 2013; 18:1261-1270). The plasma and saliva
specimens were procured before the first treatment. FIG. 4
illustrates the results from saliva and plasma showing the
respective EGFR mutation compared with biopsy-based genotyping. The
original amperometric current signals from the detection of
p.E746-A750del exon19 deletion group in saliva samples are shown in
FIG. 4E, with data was read-out at 60 seconds. The amperometric
currents of the exon 19 deletion group (p.E746-A750del) detected by
EFIRM using an exon 19 probe were significantly higher than those
in the wild-type and p.L858R mutant groups based on saliva (FIG. 4A
and FIG. 4E; 106.3.+-.13.2 in the p.E746-A750del group vs.
12.8.+-.7.5 in the wild-type group and 6.3.+-.4.7 in the p.L858R
mutant group; P<0.0001). The amperometric currents of the exon
21 mutant group (p.L858R) detected by EFIRM using the L858R probe
were significantly higher than those in the wild-type and p.L858R
mutant groups (FIG. 4B; 66.5.+-.27.2 in the p.L858R mutant group
vs. 9.5.+-.5.3 in the wild-type group and 7.7.+-.4.2 in the
p.E746-A750del group; P<0.0001). Similar results were obtained
from plasma using the probe designed for the exon 19 deletion group
(FIG. 4C; 117.2.+-.8.1 in the p.E746-A750del group vs. 20.7.+-.11.7
in the wild-type group and 10.4.+-.9.2 in the p.L858R mutant group;
P<0.0001) and for the p.L858R mutant group (FIG. 4D;
79.0.+-.34.2 in the p.L858R mutant group vs. 18.1.+-.8.9 in the
wild-type group and 13.5.+-.7.4 in the p.E746-A750del group;
P<0.0001). It is suggested that a cutoff at 2 SDs above the mean
value from the control group to differentiate the mutant and
control groups. These findings indicated that EFIRM could be used
to detect specific EGFR mutations in the plasma and saliva of
patients with NSCLC.
Blinded and Randomized Study to Detect EGFR Mutations in NSCLC
Patients Using the Platform Assay
[0121] To determine whether EGFR oncogenic mutations can be
detected in the saliva of NSCLC patients using the optimized system
technology, blinded saliva from 40 NSCLC patients harboring the two
most common EGFR tyrosine kinase domain mutations (p.L858R and exon
19 deletion) and evaluated by the system of the present
invention.
[0122] The forty saliva samples were obtained from patients with
advanced NSCLC were collected at the National Cheng Kung University
Hospital (NCKUH), blinded, tested, and sent to the University of
California, Los Angeles (UCLA) for validation. Biopsy-based EGFR
genotyping was performed at NCKUH (Table 2). The blinded samples
were further randomized by a biostatistician from a third
institution at MD Anderson Cancer Center (MDACC) followed by EFIRM
measurements of the two EGFR mutations at UCLA. The platform assay
was used to detect the two EGFR mutations in saliva of these
patients at UCLA.
[0123] The patient cohort consisted of 22 men and 18 women, with a
mean age of 58.8.+-.10.4 years; 32 cases (80%) exhibited stage IV
cancer and most patients were non-smokers (Table 1). The clinical
characteristics, including tumor stage and EGFR mutations in the
blinded group, were similar to those in the testing group.
[0124] The amperometric currents of the exon 19 deletion group
detected by EFIRM using an exon 19 probe were significantly higher
than those in the wild-type and p.L858R mutant groups (FIG. 5A;
126.6.+-.58.6 in the p.E746-A750del group vs. 14.5.+-.3.5 in the
wild-type group and 9.6.+-.3.7 in the p.L858R mutant group;
P<0.0001). Similar results were obtained using a probe designed
for the p.L858R mutant group (FIG. 4B; 113.2.+-.75.1 in the p.L858R
mutant group vs. 15.9.+-.9.6 in the wild-type group and 9.5.+-.3.2
in the p.E746-A750del group; P<0.0001). The receiver operating
characteristic analysis (FIG. 5C) indicated that the AUCs were 0.94
(95% CI, 0.82-1) and 0.96 (95% CI, 0.90-1) (FIG. 5C) for probes
carrying the p.E746-A750del or the p.L858R mutation,
respectively.
Correlation of EGFR Mutation Status Between Plasma and Saliva Using
EFIRM
[0125] The findings from the plasma were compared with those from
the saliva to evaluate whether saliva could be as informative as
plasma and serve as an additional bodily fluid for mutation
testing. For the testing group, the amperometric currents of saliva
were correlated with those from plasma using the two different
probes (FIG. 6A; R=0.98, P<0.0001 in the p.E746-A750del groups
and R=0.99, P<0.0001 in the p.L858R groups).
[0126] Similar results were observed in the blinded group (33
plasma samples). It was first demonstrated that EFIRM could be used
to detect specific EGFR mutations in the plasma of these 33
patients (FIG. 7A and FIG. 7B). As in the testing cohort, it was
found that the amperometric currents of plasma detected by EFIRM
correlated with those from saliva (FIG. 6B; R=0.94, P<0.0001 in
the p.E746-A750del groups and R=0.92, P<0.0001 in the p.L858R
groups).
TABLE-US-00001 TABLE 1 Table 1 Patient characteristics of testing
group and blinded validation group. Non-blinded cohort Blinded
validation cohort P value Age 62.1 .+-. 12.7 58.8 .+-. 10.4
0.28.sup.a Sex Total 22 40 1.00.sup.b Male 12 (54.5%) 22 (55.0%)
Female 10 (45.5%) 18 (45.0%) Smoker 7 (31.8%) 11 (27.5%) 0.95.sup.b
Stage 1.00.sup.c III 5 (22.7%) 8 (20.0%) IV 17 (77.3%) 32 (80.0%)
EGFR mutant type 0.98.sup.b Wild 11 (50.0%) 20 (50.0%) L858R 7
(31.8%) 12 (30.0%) Exon 19del 4 (18.2%) 8 (20.0%) .sup.aT test;
.sup.bchi-square test; .sup.cFisher's test
TABLE-US-00002 TABLE 2 Table 2 Clinical characteristics and
treatment course of four patients receiving TKI Duration Best EGFR
of TKI use clinical Age Sex Stage mutation TKI use (days) response
Current treatment Case 1 68 M IV Exon 19 Afatinib 391 PR Alimta +
cisplatin Del Case 2 49 M III Exon 19 Iressa 335 SD Alimta +
cisplatin Del Case 3 77 F IV Exon 19 Iressa 222 PR Iressa Del Case
4 58 F IV Exon 21 Iressa 103 SD Alimta + cisplatin L858R F, female;
M, male; PR, partial response; SD, stable disease
[0127] The amperometric currents of exon19 deletion group detected
by EFIRM using an exon19 probe were significantly higher than those
in the wild type and p.L858R mutant groups (FIG. 5A, 126.6.+-.58.57
in p.E746-A750del group versus 14.52.+-.3.54 in wild type group and
9.61.+-.3.67 in p.L858R mutant group, p<0.0001) Similar results
are obtained using the probe designed for p.L858R mutant group
(FIG. 5B, 113.2.+-.75.06 in p.L858R mutant group versus
15.85.+-.9.65 in wild type group and 9.54.+-.3.24 in p.E746-A750del
group, p<0.0001). Receiver operating characteristic analysis
(FIG. 5C) indicated that for probes carrying the p.E746-A750del or
the p.L858R mutation, the areas under the curve (AUCs) were 0.94
(95% CI, 0.82 to 1) and 0.96 (95% CI, 0.90 to 1) (FIG. 5C; Table
3), respectively. (Table 3).
TABLE-US-00003 TABLE 3 Table 3 Receiver operating characteristic
analysis of TKI treatment. Lower Upper Explanation AUC CI CI
AUC_Ex19Del_excluding_TKI 0.9414 0.8245 1
AUC_Ex19Del_w_TKI_as_wildtype 0.9375 0.8133 1
AUC_L858R_excluding_TKI 0.9583 0.8994 1 AUC_L858R_w_TKI_as_wildtype
0.9505 0.882 1 AUC_both_Probe_excluding_TKI 0.9325 0.8538 1
AUC_both_Probe_w_TKI_as_wildtype 0.9229 0.8356 1 CI, confidence
interval.
The System of the Present Invention for Oncogene Mutation
Analysis
[0128] Current oncogene mutation detection technologies mainly
PCR-based, requiring sample pretreatment and several hours of
detection. Via the system of the present invention, EGFR mutations
were accurately identified from circulating EGFR sequences. The
system exploits (1) simple and effective biomarker release from
body fluids, (2) enhanced sample mixing and accumulation, (3)
enhanced hybridization of the EGFR gene, and (4) suppression of
non-specific interference. When exposed to a non-uniform electrical
field, DNA/RNA is rapidly released in situ. The specificity of each
mutation is expected to yield a unique electric-field profile in
terms of voltage, cycle numbers, duration, and other parameters.
Positive potential in the electric field (cyclic square wave)
facilitates accumulation of the gene target onto the working
electrode, while negative potential removes weakly bound
non-specific sequences. In addition to enhancing hybridization, the
cyclic square wave electric field also generates near-field
solution mixing and accumulation, due to the continuous flapping of
the electrical field. The system only permits perfectly matched
sequence hybridization; mismatched sequences are removed. By
individually optimizing the electric profile for each target
sequence of interest, the system achieves sensitivity and
specificity that are comparable with those of quantitative PCR,
while only requiring a few microliters of the clinical sample.
[0129] Currently it is not entirely clear how mechanistically
tumor-specific oncogenic mutations are detected in saliva. In a
previous study, it was demonstrated that breast cancer-derived
microvesicles are capable of interacting with salivary gland cells,
altering the composition of their secreted microvesicles (Lau C S,
Wong D T. Breast Cancer Exosome-like Microvesicles and Salivary
Gland Cells Interplay Alters Salivary Gland Cell-Derived
Exosome-like Microvesicles In Vitro. PLoS ONE 2012; 7: e33037);
inhibiting exosome biogenesis in pancreatic cancer results in the
ablation of the development of discriminatory salivary biomarkers
(Lau C, Kim Y, Chia D, et al. Role of Pancreatic Cancer-derived
Exosomes in Salivary Biomarker Development J Biol Chem 2013; 288:
26888-26897). Recent investigations provide further evidence that
exosomes carry not only protein, RNA, microRNA, mitochondrial DNA
(Guescini M, Guidolin D, Vallorani L, et al. C2C12 myoblasts
release micro-vesicles containing mtDNA and proteins involved in
signal transduction. Experimental cell research 2010; 316:
1977-1984), and single-stranded DNA, but also large fragments of
double-stranded mutated Kras and p53 DNA (Kahlert C, Melo S A,
Protopopov A, et al. Identification of Double Stranded Genomic DNA
Spanning all Chromosomes with Mutated KRAS and p53 DNA in the Serum
Exosomes of Patients with Pancreatic Cancer. The Journal of
biological chemistry 2014). More importantly, tumor-specific
EGFRvIII was previously detected in serum microvesicles (Paez J G,
Janne P A, Lee J C, et al. EGFR mutations in lung cancer:
correlation with clinical response to gefitinib therapy. Science
2004; 304: 1497-1500) and was shown to be released into the blood
to merge with the plasma membranes of cancer cells lacking EGFRvIII
(Al-Nedawi K, Meehan B, Micallef J, et al. Intercellular transfer
of the oncogenic receptor EGFRvIII by microvesicles derived from
tumor cells. Nature cell biology 2008; 10: 619-624), it is
reasonable to hypothesize that lung cancer cell-delivered
microvesicles carrying EGFR DNA affect the DNA content of other
cells, such as those in the salivary glands. However, the
mechanisms by which lung-cancer cells release these microvesicles
and disperse them via the blood to distant sites remain to be
established.
The Influence of TKI Treatment on Detection of EGFR Mutations
[0130] During analysis of the correlation of detection of EGFR
mutations in saliva from NSCLC patients, it was realized that four
patients had previously undergone treatment with TKIs prior to
enrolling in the study (Table 2). The amperometric currents of
these four samples were significantly lower (10.about.25-nA) than
the signal from NSCLC patients with the same EGFR mutations but no
prior treatment with TKIs. The signals from the TKI-treated cases
were similar to those patients with wildtype EGFR. It was
hypothesized that TKI treatment effectively suppress the growth of
NSCLC cells, leading to less tumor shedding of circulating tumor
cells and/or shedding of mutated EGFR (free form or exosome bound),
and effectively lesser detection by the system of the present
invention in saliva. This is congruent with the present data. It
should be noted that data analysis excluded these 4 TKI previously
treated cases.
Current Clinical Practice for Detecting EGFR Mutations in NSCLC
Patients
[0131] Direct sequencing of amplified DNA products is the most
popular method for detecting EGFR mutations. This strategy often
takes up to 3-4 weeks to yield results, and is clinically limited
by low sensitivity and false negative or non-informative results,
especially for cytology specimens. Several new techniques,
including the use of TaqMan PCR, and denaturing high-performance
liquid chromatography have been introduced (Janne P, Borras A,
Kuang Y, et al. A rapid and sensitive enzymatic method for
epidermal growth factor receptor mutation screening. Clinical
cancer research: an official journal of the American Association
for Cancer Research 2006; 12: 751-758; Chin T, Anuar D, Soo R, et
al. Detection of epidermal growth factor receptor variations by
partially denaturing HPLC. Clin Chem 2007; 53: 62-70; Endo K,
Konishi A, Sasaki H, et al. Epidermal growth factor receptor gene
mutation in non-small cell lung cancer using highly sensitive and
fast TaqMan PCR assay. Lung Cancer 2005; 50: 375-384), but none
have been adopted as a standard clinical method for detecting EGFR
mutations.
[0132] In addition, practical clinical obstacles often exist
pertaining to the acquisition and availability of appropriate
tissue samples as well as intratumoral genetic heterogeneity. In
patients with advanced NSCLC, tumor tissue is not always available
for EGFR mutation testing, either because only small amounts of
tissue are collected or because the collected tissues have very
low, or no, levels of tumor, according to CT-guided or
bronchoscopic biopsy. Recent evidence suggests that regionally
separated heterogeneous somatic mutational events can lead to
sampling bias, which impairs the interpretation of genomics data
derived from single-tumor biopsies (Gerlinger M, Rowan A J,
Horswell S, et al. Intratumor heterogeneity and branched evolution
revealed by multiregion sequencing. The New England journal of
medicine 2012; 366: 883-892; Yap T A, Gerlinger M, Futreal P A,
Pusztai L, Swanton C. Intratumor heterogeneity: seeing the wood for
the trees. Science translational medicine 2012; 4: 127ps110). For
patients who do not have enough tumor tissue for a mutation assay,
or who are suspected of being associated with a false-negative
result due to tumor heterogeneity within a tissue sample, body
fluids-based EGFR mutation detection may substitute for tissue
biopsy for molecular diagnosis.
Clinical Applications of the System of the Present Invention
[0133] Applying the system of the present invention to directly
detect EGFR mutations from saliva of NSCLC patients will provide
information on EGFR mutation status in a non-invasive, rapid and
cost effective. These advantages will enable clinicians to adjust
their therapeutic strategies in a timely fashion, consequently
improving the clinical outcome of EGFR-targeting therapy. Since the
system is based on an electrochemical platform, it can be easily
transformed into high-throughput oncogenic mutation analysis lab
assays as well as point-of-care devices for rapidly identifying
oncogenic mutations on site. A portable point-of-care device that
uses saliva samples for cancer detection based on this platform has
already been developed (data and prototype not shown). In
additional to assisting treatment decision making, the system also
has the potential to diagnosis or screen for lung cancer. For
patients who do not have enough tumor tissue for a mutation assay,
mutation detection via the present invention may substitute for
tissue biopsy. Another impactful application of the system
technology is to address the well-known drug resistance eventually
develops during the course of treatment with these EGFR-TKIs as a
result of secondary mutations in the EGFR kinase domain, hampering
the overall improvement in survival following EGFR-targeting
treatment. The present invention is a non-invasive approach that
will permit continuous monitoring of somatic EGFR mutations during
lung-cancer progression. Thirdly, for patients with lung nodule,
the platform will be helpful in differential diagnosis. Finally,
the system has the potential to screen for lung cancer,
particularly in people who have never smoked. Although most lung
cancers result from smoking, .about.25% of lung-cancer cases
worldwide occur in people who have never smoked, accounting for
>300,000 deaths each year (Sun S, Schiller J H, Gazdar A F. Lung
cancer in never smokers--a different disease. Nature reviews Cancer
2007; 7: 778-790; Wakelee H A, Chang E T, Gomez S L, et al. Lung
cancer incidence in never smokers. Journal of clinical oncology:
official journal of the American Society of Clinical Oncology 2007;
25: 472-478). Following consideration of the amount of exposure to
cigarette smoke, low-dose CT-based examination has been proposed
for high-risk subjects to provide an early diagnosis of lung cancer
(Boiselle P M. Computed tomography screening for lung cancer. JAMA:
the journal of the American Medical Association 2013; 309:
1163-1170). For people who have never smoked, however, no ideal
method has been established to detect those at high risk of cancer.
EGFR mutations are the first specific genetic mutations to be
associated with cancer in patients who have never smoked, and
increasing smoke exposure is negatively correlated with EGFR
mutation status (Pham D, Kris M G, Riely G J, et al. Use of
cigarette-smoking history to estimate the likelihood of mutations
in epidermal growth factor receptor gene exons 19 and 21 in lung
adenocarcinomas. Journal of clinical oncology: official journal of
the American Society of Clinical Oncology 2006; 24: 1700-1704).
Compared to blood sampling for detecting circulating DNA,
collecting saliva is non-invasive, and therefore using the present
invention to detect EGFR mutations will be advantageous to
blood-based diagnosis in the screening of early lung cancers,
especially in people who have never smoked.
[0134] The disclosures of each and every patent, patent
application, and publication cited herein are hereby incorporated
herein by reference in their entirety.
[0135] While this invention has been disclosed with reference to
specific embodiments, it is apparent that other embodiments and
variations of this invention may be devised by others skilled in
the art without departing from the true spirit and scope of the
invention. The appended claims are intended to be construed to
include all such embodiments and equivalent variations.
Sequence CWU 1
1
4122DNAArtificial SequenceChemically Synthesized 1tgttgcttcc
ttgatagcga cg 22221DNAArtificial SequenceChemically Synthesized
2ggaattttaa ctttctcacc t 21321DNAArtificial SequenceChemically
Synthesized 3cagtttggcc cgcccaaaat c 21422DNAArtificial
SequenceChemically Synthesized 4ttgacatgct gcggtgtttt ca 22
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