U.S. patent application number 15/133996 was filed with the patent office on 2018-01-04 for molecular analysis using a magnetic sifter and nanowell system.
The applicant listed for this patent is The Board of Trustees of the Leland Stanford Junior University. Invention is credited to Sanjiv Sam Gambhir, Viswam S. Nair, Chin Chun Ooi, Seung-min Park, Shan X. Wang, Dawson Wong.
Application Number | 20180002760 15/133996 |
Document ID | / |
Family ID | 60813546 |
Filed Date | 2018-01-04 |
United States Patent
Application |
20180002760 |
Kind Code |
A2 |
Park; Seung-min ; et
al. |
January 4, 2018 |
Molecular Analysis using a Magnetic Sifter and Nanowell System
Abstract
A method for identification of circulating tumor cells (CTCs) in
a blood sample uses magnetic enrichment and a nanowell assay. The
CTCs are magnetically labeled with cancer cell markers conjugated
to magnetic nanoparticles and then separated by passing the blood
sample through a magnetic sifter. The enriched CTCs are then loaded
into a microfluidic single-cell molecular assay comprising an array
of 25,600 or more nanowells, each containing at most a single one
of the CTCs. Using multiple fluorescent gene markers, simultaneous
multiple-color multiplexed gene expression of the CTCs is
performed, preferably using RT-PCR. Images of fluorescence signals
from individual nanowells are analyzed to identify CTCs.
Inventors: |
Park; Seung-min; (Menlo
Park, CA) ; Wong; Dawson; (San Jose, CA) ;
Ooi; Chin Chun; (Menlo Park, CA) ; Gambhir; Sanjiv
Sam; (Portola Valley, CA) ; Nair; Viswam S.;
(Menlo Park, CA) ; Wang; Shan X.; (Portola Valley,
CA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
The Board of Trustees of the Leland Stanford Junior
University |
Palo Alto |
CA |
US |
|
|
Prior
Publication: |
|
Document Identifier |
Publication Date |
|
US 20160230237 A1 |
August 11, 2016 |
|
|
Family ID: |
60813546 |
Appl. No.: |
15/133996 |
Filed: |
April 20, 2016 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
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62149978 |
Apr 20, 2015 |
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62150100 |
Apr 20, 2015 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C12Q 2521/107 20130101;
C12Q 2537/143 20130101; C12Q 1/686 20130101; C12Q 2600/158
20130101; G06F 16/134 20190101; G06F 16/172 20190101; G06F 12/0802
20130101; G06F 2212/225 20130101; C12Q 1/6886 20130101; C12Q 1/686
20130101 |
International
Class: |
C12Q 1/68 20060101
C12Q001/68 |
Goverment Interests
STATEMENT OF GOVERNMENT SPONSORED SUPPORT
[0002] This invention was made with Government support under
contract CA151459 and CA185804 awarded by the National Institutes
of Health. The Government has certain rights in the invention.
Claims
1. A method for identification of circulating tumor cells (CTCs),
the method comprising: magnetically labeling the CTCs in a blood
sample with cancer cell markers conjugated to magnetic
nanoparticles; separating the magnetically labeled CTCs by passing
the blood sample through a magnetic sifter during application of an
external magnetic field; collecting the separated magnetically
labeled CTCs to produce enriched CTCs; loading the enriched CTCs
into a microfluidic single-cell molecular assay comprising an array
of 25,600 or more nanowells, where each of the nanowells is adapted
to contain at most a single one of the CTCs; performing multiple
simultaneous multiple-color multiplexed gene expression of the CTCs
using the microfluidic single-cell molecular assay and multiple
fluorescent gene markers; imaging the array of nanowells using
fluorescence signal acquisition from individual nanowells,
producing images of the array of nanowells; analyzing the images
using a signal processor to identify CTCs based on the concurrent
expression of two or more genes.
2. The method of claim 1 further comprising: performing multiple
simultaneous multiple-color multiplexed genotyping of the CTCs for
mutational detection using the microfluidic single-cell molecular
assay and multiple fluorescent gene markers.
3. The method of claim 1 further comprising: performing multiple
simultaneous multiple-color multiplexed genotyping of the CTCs
wherein non-CTCs are identified based on the intensity of gene
expression of one or more genes.
4. The method of claim 1 wherein the microfluidic single-cell
molecular assay is performed by RT-PCR.
5. The method of claim 1 wherein the cancer cell markers are
conjugated to magnetic nanoparticles through one or more molecules
selected from epithelial cell adhesion molecule antibodies, HER2
antibodies, and other antibodies against cancer surface
markers.
6. The method of claim 1 further comprising: double sifting of the
magnetically labeled CTCs, performing red blood cell lysis,
performing DNase treatment, and performing CD45 staining for
leukocyte exclusion.
7. The method of claim 1 wherein analyzing the images using a
signal processor to identify CTCs comprises using an outlier
identification method such that a nanowell is classified as
positive if its expression of two or more genes is greater than 3
standard deviations away from a distribution expected for empty
wells.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application claims priority from U.S. Provisional
Patent Application 62/149,978 filed Apr. 20, 2015, and from U.S.
Provisional Patent Application 62/150,100 filed Apr. 20, 2015, both
of which are incorporated herein by reference.
FIELD OF THE INVENTION
[0003] The present invention relates generally to biomedical
sensing techniques. More particularly, the invention relates to
systems and methods for identifying and classifying circulating
tumor cells.
BACKGROUND OF THE INVENTION
[0004] Circulating tumor cells (CTCs), shed from a primary tumor
into the bloodstream, may be valuable diagnostic/prognostic
biomarkers that contain actionable genetic information for tumor
analysis. Unfortunately, the rarity of CTCs in comparison to other
blood components necessitates high-throughput separation
technologies for efficient enrichment and elaborate downstream
analysis. Moreover, genetic data extraction from CTCs currently
suffers from a lack of reliable analytical methods capable of
handling a low number of cells. Urgent needs in technological
support require developing new diagnostic platforms that can either
detect cancer at an early stage, where cancer cells may be more
difficult to detect, or monitor tumor progression.
SUMMARY OF THE INVENTION
[0005] The present invention provides a method for obtaining gene
expression profiles of individual CTCs for cancer diagnosis and/or
prognosis. The invention also provides an integrated CTC platform
for implementing the method. The present invention provides
capability to clearly distinguish between cells of cancer and
non-cancer origins, with higher sensitivity and specificity than
that of the state-of-the-art immunostaining classification
technique of CTC identification.
[0006] In one aspect, the invention provides a method for
identification of circulating tumor cells (CTCs) in a blood sample
using magnetic enrichment and a nanowell assay. The CTCs are
magnetically labeled with cancer cell markers conjugated to
magnetic nanoparticles. For example, the cancer cell markers may be
conjugated to magnetic nanoparticles through epithelial cell
adhesion molecule antibodies, HER2 antibodies, or other antibodies
against cancer surface markers. The magnetically labeled CTCs are
then separated by passing the blood sample through a magnetic
sifter during application of an external magnetic field. The
separated magnetically labeled CTCs are then collected to produce
enriched CTCs, which are then loaded into a microfluidic
single-cell molecular assay comprising an array of 25,600 or more
nanowells, where each of the nanowells is adapted to contain at
most a single one of the CTCs. Using the microfluidic single-cell
molecular assay and multiple fluorescent gene markers, multiple
simultaneous multiple-color multiplexed gene expression of the CTCs
is performed, preferably using RT-PCR, where there is concurrent
expression of two or more genes. The array of nanowells is imaged
using fluorescence signal acquisition from individual nanowells,
producing images of the array of nanowells. The images are analyzed
using a signal processor to identify CTCs based on the concurrent
expression of two or more genes. Preferably, the image analysis
identifies CTCs using an outlier identification method such that a
nanowell is classified as positive if its expression of two or more
genes is greater than 3 standard deviations away from a
distribution expected for empty wells.
[0007] The method may include one or more additional steps to
further enhance sensitivity, such as double sifting of the
magnetically labeled CTCs, performing red blood cell lysis,
performing DNase treatment, or performing CD45 staining for
leukocyte exclusion.
[0008] The method may include performing multiple simultaneous
multiple-color multiplexed genotyping of the CTCs for mutational
detection using the microfluidic single-cell molecular assay and
multiple fluorescent gene markers. The method may include
performing multiple simultaneous multiple-color multiplexed
genotyping of the CTCs where non-CTCs are identified based on the
intensity of gene expression of one or more genes.
[0009] These and other aspects of the invention are set forth in
more detail in the following detailed description and associated
drawing figures.
BRIEF DESCRIPTION OF THE DRAWINGS
[0010] FIG. 1 illustrates a device integrating a magnetic
enrichment chamber and detection chamber for implementing a method
for identification of circulating tumor cells (CTCs) in a blood
sample, according to an embodiment of the invention.
[0011] FIGS. 2A, 2B, 2C illustrate three steps in a technique for
separating magnetically labeled CTCs from other cells using a
magnetic sifter, according to an embodiment of the invention.
[0012] FIG. 2D illustrates a step of seeding a nanowell array with
CTCs, according to an embodiment of the invention.
[0013] FIG. 2E illustrates a step of performing simultaneous
multiple-color multiplexed gene expression of the CTCs in the
nanowell array, according to an embodiment of the invention.
[0014] FIG. 2F shows an image of fluorescence signal acquisition
from individual nanowells where cMET is expressed in particular
individual nanowells, according to an embodiment of the
invention.
[0015] FIG. 2G shows an image of fluorescence signal acquisition
from individual nanowells where hTERT is expressed in particular
individual nanowells, according to an embodiment of the
invention.
[0016] FIG. 3A shows the results of four-color gene expression,
where white blood cells only express VIM significantly, according
to an embodiment of the invention.
[0017] FIG. 3B shows the results of four-color gene expression,
where H1650 cells fully express hTERT, cMET, and VIM, and partially
express ALDH1A3, according to an embodiment of the invention.
[0018] FIG. 3C is a graph of an ensemble analysis showing the clear
discernment of TERT/MET expression levels in WBC and H1650 cells,
according to an embodiment of the invention.
[0019] FIGS. 4A, 4B show the bulk RT-PCR signal intensity versus
the thermal cycle number for two cell lines (HCC827 and H661) used
to represent different gene expression patterns according to their
Epidermal Growth Factor Receptor (EGFR) Exon 19 deletion mutational
status, according to an embodiment of the invention.
[0020] FIGS. 4C, 4D are nanowell images of the same two cell lines
(HCC827 and H661) spiked into a nanowell device, according to an
embodiment of the invention.
[0021] FIG. 5 is a graph comparing the number of nanowells with
double positive (hTERT+/cMET+) expression of five healthy control
individuals with a NSCLC patient (stage undetermined), a recurrent
NSCLC patient, multiple NSCLC stage I/II patients, and multiple
NSCLC stage IV patients, according to an embodiment of the
invention.
DETAILED DESCRIPTION
[0022] Embodiments of the present invention include integrated
nanotechnology methods and devices for biomedical analysis that
enable the molecular profiling of tumor-derived cells from
peripheral blood samples.
[0023] In one embodiment of the invention, shown in FIG. 1, a
device provides a technological integration of a magnetic
enrichment chamber 100 and a detection chamber 102.
[0024] The magnetic enrichment chamber 100 is a magnetic rare cell
separation platform for isolating and enriching rare cells from
peripheral blood samples. Preferably, it includes a magnetic sifter
(MagSifter.TM.) device. This magnetic sifter offers increased
capture efficiency at high flow rates due to extreme field
gradients at the pore edges, high throughput due to the density of
pores (.about.200 pores/mm.sup.2), scalability via standard
lithographic fabrication, and harvesting of viable cells. The
inlets 106 allow injection of blood samples or reagents, while
unwanted cells and blood components as well as wastes are collected
at outlet 108. The eluted CTCs are passed to detection chamber
through the connecting channel 110.
[0025] The detection chamber 102 is a multiplexed microfluidic
device that performs single-cell RT-PCR of the isolated rare cells
using a nanowell array assay. This microfluidic single-cell
molecular assay performs multiple simultaneous gene expression
analysis and genotyping from single cells. Specific gene markers
are selected for (a) early diagnosis of cancer, and (b) genotyping,
e.g. mutational analysis, which may inform cancer therapy selection
and monitoring. The inlets 112 allow injection of lysis buffer and
RT-PCR reagents, and the excess reagents and wastes are passed to
outlet 114.
[0026] The detection chamber 102 includes a massive multiplexed
array of nanowells that enables single-cell RT-PCR on chip of rare
cells that have been isolated by a magnetic sifter device from
whole blood samples. In one embodiment, a standard optical and
signal processing module 104 is used to interrogate the Nanowell
using conventional fluorescence microscopy to detect a candidate
panel of genes on CTCs that are relevant for cancer detection or
therapy monitoring. The nanowell is low-cost (PDMS-based) and
scalable (from currently 25 k to more than 100 k nanowells). The
nanowell-based RT-PCR has sufficient efficiency for the
discrimination of cell sub-populations. One to thousands of cells
that have been enriched by the Magnetic sifter cell sorters can be
loaded into the nanowell by direct pipetting and centrifugation
(e.g., 3,000 rpm for 10 min.) or by automated fluidics techniques.
The nanowell dimensions are engineered such that single cells
settle into different wells, each of which serves as a miniaturized
RT-PCR reaction chamber for mRNA analysis. For example, in one
implementation, each of the 25,600 nanowell compartments are
20.times.20.times.50 m.sup.3, with 20-pL volumes that are 10.sup.6
times smaller than those of typical PCR assays, thereby enabling
higher sensitivity from higher mRNA concentrations.
[0027] In another embodiment, a four-color-based single-cell gene
expression assay is used to classify each captured cell from a
patient sample. Isolation and enrichment of CTCs from the magnetic
sifter is assayed by the nanowell for massive single-cell targeted
gene expression analysis. This is typically achieved within 8 hours
from patient blood draw to data analysis.
[0028] Various techniques may be used to improve integration of
these two technological platforms for better mutual compatibility
and to enable high-sensitivity and high-specificity molecular
profiling of tumor-derived cells. For example, these techniques may
include double sifting, red blood cell lysis, deoxyribonuclease
(DNase) treatment, and CD45 staining for leukocyte exclusion. For
example, double sifting can be performed by collecting the eluted
cells at the outlet 108 and re-sifting in chamber 100 before
passing the final eluted cells to the detection chamber 102.
[0029] A method for identification of circulating tumor cells
(CTCs) in a blood sample using magnetic enrichment and a nanowell
assay will now be described in more detail. The method effectively
enriches rare cells via a magnetic sifting technology using
magnetic nanoparticles to tag CTCs in conjunction with magnetic
filtration to enable high-throughput enrichment with release
capability. For subsequent characterization of the enriched cells,
a robust microwell-based assay was designed to circumvent
experimental errors associated with ensemble measurements through
detection of mRNA transcripts directly from single CTCs (using
one-step RT-PCR). These massive single-cell arrays are able to
isolate up to thousands of single lung cancer cells to measure gene
expression and to observe the translational kinetics of single
cancer cells.
[0030] As an initial step, CTCs in a whole blood sample (e.g., 2
mL) are magnetically labeled with cancer cell markers conjugated to
magnetic nanoparticles. For example, the cancer cell markers may be
epithelial cell-adhesion molecules (EpCAM), Human Epidermal
[0031] Growth Factor Receptor 2 (HER2) antigens, or other cancer
surface markers. In one embodiment, streptavidin coated 150-nm iron
oxide magnetic nanoparticles (R&D, MAG999) are conjugated to
biotinylated anti-EpCAM antibodies (BioLegend) which will in turn
couple to the EpCAM markers on the cancer cells.
[0032] The magnetically labeled CTCs are then separated by pumping
the blood sample through pores of a magnetic sifter during
application of an external magnetic field. FIG. 2A shows labeled
CTCs 120 and other cells 122 before passing through a sifter 124.
FIG. 2B shows the labeled CTCs 120 attached to the edges of the
pores of the sifter 124 during application of a magnetic field H,
while the other (unlabeled) cells 122 pass freely through the
pores. This separates the CTCs from the other cells. FIG. 2C shows
the labeled CTCs 120 released from the edges of the pores of the
sifter 124 after the magnetic field H is turned off, allowing the
separated CTCs 120 to be released and collected for downstream
analysis.
[0033] After the separated magnetically labeled CTCs are collected
to produce enriched CTCs, they are then loaded into a microfluidic
single-cell molecular assay comprising an array 140 of 25,600 or
more nanowells, as shown in FIG. 2D. Each of the nanowells in the
array 140 is adapted to contain only a single one of the CTCs or to
remain empty.
[0034] For example, in one implementation, after cell collection,
the effluent from the device is subsequently treated with red blood
cell lysis buffer (ammonium chloride-based) to further remove RBC
contamination, and also treated with DNase to remove all possible
DNA fragments of non-CTC origin. Optionally, the effluent may be
sent through the magnetic sifter again in order to increase
purity.
[0035] All effluent is then loaded on top of a nanowell device by
centrifugation, and cellular contents are seeded into individual
nanowell compartments. Preferably, fluorescence microscope images
of the entire nanowell array are acquired for identification and
exclusion of wells containing WBCs by CD45 signal. After drying
(e.g., 70.degree. C. for 10 min) to fix seeded cells into the wells
and to completely deactivate the DNase, single-cell RT-PCR master
mix is applied to the nanowells, which are then sealed with a small
piece of adhesive PCR sealant film (Bio-Rad). In one
implementation, the RT-PCR master mix consisted of 2.times.
reaction mix (CellsDirect.TM. One-Step qRT-PCR, Life Technologies),
polymerases (SuperScript.RTM. III RT/Platinum.RTM. Taq Mix),
TaqMan.RTM. probes (Life Technologies; Bio-Rad, Hercules, Calif.)
for targeting specific genes, and DEPC-treated water
[0036] After the seeding and reagent application, gene expression
of the CTCs in each individual nanowell is performed. More
specifically, the microfluidic single-cell molecular assay and
multiple fluorescent gene markers are used to perform multiple
simultaneous multiple-color multiplexed gene expression of the
CTCs. This is preferably performed using RT-PCR, where there is
concurrent expression of two or more genes in each individual
nanowell, as shown in FIG. 2E. For example, in one implementation,
the nanowell chip is placed into a thermocycler (PTC-200, Peltier
Thermal Cycler, Bio-Rad) for gene expression via PCR amplification
using the following cycle parameters: for the first thermal cycler
step, cell lysis and subsequent reverse transcription, the array
was incubated at 50.degree. C. for 45 min. This was followed by 10
cycles of 60 s at 95.degree. C. for denaturation and 90 s at
65.degree. C. for an annealing and extension step. Amplification
commenced after with 35 cycles of 60 s at 90.degree. C. and 90 s at
60.degree. C. For fully automatic work flow, a heating element (not
shown) is integrated in the detection chamber to facilitate thermal
cycling.
[0037] The array of nanowells is imaged using fluorescence
microscope signal acquisition from individual nanowells, producing
images of the array of nanowells.
[0038] For example, FIG. 2F shows signal acquisition from
individual nanowells where cMET is expressed in particular
individual nanowells, and FIG. 2G shows signal acquisition from
individual nanowells where hTERT is expressed in particular
individual nanowells. Single CTCs displaying hTERT only, cMet only,
and both were evident upon imaging.
[0039] Because each nanowell contains at most one CTC, the image
signals from the individual nanowells allow identification of CTCs
on an individual cell basis.
[0040] The images are analyzed using a signal processor to identify
CTCs based on the concurrent expression of two or more genes. The
individual nanowell fluorescence signals are analyzed for
identification of tumor-derived cellular material based on double
positive (cMET+ and hTERT+) gene expression. Preferably, the image
analysis identifies CTCs using an outlier identification method
such that a nanowell is classified as positive if its expression of
two or more genes is greater than 3 standard deviations away from a
distribution expected for empty nanowells. This outlier
identification method capitalizes on the fact that the majority of
the wells are empty due to the rarity of the putative CTCs.
Assuming a Gaussian distribution for the empty well signals, we
define wells as being positive if they are greater than 3 standard
deviations away from the distribution expected for these empty
wells.
[0041] Since each Nanowell array contains 25,600 wells and the CTC
population from 2 mL of blood is expected to be no more than 2,000
cells, each individual well has a 99.7% chance of containing either
a single cell or no cell, according to the Poisson distribution,
and only a 0.3% chance of containing two or more cells, thereby
representing a high-throughput method of analyzing "single"
cells.
[0042] The nanowell assay provides significant advantages over
conventional bulk assay. In a bulk assay (RT-PCR of either from all
cells from cell culture or all the cells captured by magnetic
separation), the background signal is inevitable and a serious
limiting factor (contributing to false positives). In a nanowell
assay, the vast majority of nanowells which contribute just
background signal are automatically excluded, greatly boosting the
biological signal to noise ratio when we process the fluorescent
images.
[0043] In another embodiment, four-color gene expression from a
single nanowell can be achieved as follows. H1650 cells are spiked
into healthy whole blood samples (mimicking a patient blood
condition) and processed through the previously described workflow.
Four genes are selected for identification of CTCs:
[0044] hTERT--human telomerase reverse transcriptase;
[0045] cMET--hepatocyte growth factor receptor, a proto-oncogene)
and assessment of metastatic capability;
[0046] VIM--Vimentin, an Epithelial-to-Mesenchymal (EMT)
marker;
[0047] ALDH1A3--aldehyde dehydrogenase.
[0048] Four fluorophores are used for multiplex single-cell gene
expression profiling, where primer-probe sets have four discrete
excitation-emission spectra that can be resolved by fluorescence
microscopy. Probes with 4 different fluorophores and minimal
spectral overlap are selected for simultaneous 4-plex gene
expression capability. The 4 fluorophores, FAM, HEX, Texas Red, and
Cy5, have excitation and emission peaks of 492 and 517 nm, 530 and
556 nm, 596 and 615 nm, and 650 and 670 nm, respectively.
Primer-probe assays were obtained commercially: TERT (Life
Technologies), MET (Life Technologies), VIM (Bio-Rad), and ALDH
(Bio-Rad). The 4-plex RT-PCR process is optimized on conventional
bulk assay in a CFX96 Touch.TM. Real-Time PCR Detection System
(Bio-Rad).
[0049] FIGS. 3A-C show the results of four-color gene expression
from a single nanowell. FIG. 3A shows an image of a nanowell array,
where white blood cells only express VIM significantly. FIG. 3B
shows an image of a nanowell array, where H1650 cells fully express
hTERT, cMET, and VIM, and partially express ALDH1A3 (in contrast to
white blood cells, which only express VIM significantly). FIG. 3C
is a graph of an ensemble analysis showing the clear discernment of
TERT/MET expression levels in WBC and H1650 cells.
[0050] The 4-plex assay developed to analyze CTC gene expression
can be correlated with patient diagnosis and CT and PET-CT imaging
data.
[0051] The demonstrated number multiplexed gene markers is limited
only by the fluorescent dye colors developed.
[0052] In other embodiments, multiplexed RT-PCR may be performed in
a nanowell array with other labels like Raman labels, which may
allow deep multiplexing well beyond four gene markers. For example,
Raman labels based on composite organic-inorganic nanoparticles
(COINs) are capable of multiplexed labeling well beyond four unique
optical signatures.
[0053] In another embodiment, genotyping of cells can also be
performed. For example, two NSCLC cell lines may be used to
represent different gene expression patterns according to their
Epidermal Growth Factor Receptor (EGFR) Exon 19 deletion mutational
status. Bulk PCR analysis shows that HCC827 has measured signal for
only the EGFR Exon 19 deletion mutation, while H661 exhibits
measured signal for only the EGFR wild-type gene. The same two cell
lines (HCC827 and H661) were then spiked into a nanowell device,
and the bulk PCR results can be directly translated to the
corresponding nanowell signal. HCC827 shows only the green signal
(denoting FAM dye resulting from EGFR Exon 19 deletion mutation
amplification), while H661 exhibits only the orange signal
(denoting HEX dye resulting from EGFR wild-type amplification).
These results can be applied to patient samples in order to perform
genotyping that can inform therapy selection and monitoring in
clinical settings.
[0054] FIGS. 4A-D compare genotyping in bulk vs. nanowell. In the
bulk, FIGS. 4A-B show the bulk RT-PCR signal intensity versus the
thermal cycle number for two cell lines used to represent different
gene expression patterns according to their Epidermal Growth Factor
Receptor (EGFR) Exon 19 deletion mutational status. Bulk PCR
analysis shows that HCC827 has measured signal for only the EGFR
Exon 19 deletion mutation, while H661 exhibits measured signal for
only the EGFR wild-type gene. FIGS. 4C-D are nanowell images of the
same two cell lines (HCC827 and H661) spiked into a nanowell
device, and the results from FIGS. 4A-B can be directly translated
to the corresponding Nanowell signal. HCC827 shows only the green
signal (denoting FAM dye resulting from EGFR Exon 19 deletion
mutation amplification), while H661 exhibits only the orange signal
(denoting HEX dye resulting from EGFR wild-type amplification).
These results can be applied to patient samples in order to perform
genotyping, e.g., mutational detection, that can inform therapy
selection and monitoring in clinical settings, according to the
current invention.
[0055] FIG. 5 is a graph comparing the number of nanowells with
double positive (hTERT+/cMET+) expression of five healthy control
individuals with a NSCLC patient (stage undetermined), a recurrent
NSCLC patient, multiple NSCLC stage I/II patients, and multiple
NSCLC stage IV patients. The cancer patients have generally greater
number of putative CTCs than healthy controls, indicating that our
methodology is capable of discriminating cancer patients from
health individuals.
[0056] In some embodiments, an optimized single-cell RT-PCR
procedure may be used when performing simultaneous molecular
profiling of individual cells with four genes. Specifically, the
selection of gene markers for early diagnosis and clinical
assessment of cancer includes human telomerase reverse
transcriptase (hTERT), hepatocyte growth factor receptor (cMET or
HGFR, a proto-oncogene), Vimentin (VIM, an
Epithelial-to-Mesenchymal (EMT) marker), and aldehyde dehydrogenase
(ALDH1A3). The selection of gene markers for genotyping may also
include hepatocyte growth factor receptor (cMET or HGFR, a
proto-oncogene), Epidermal Growth Factor Receptor (EGFR) Exon 19
deletion mutation, EGFR wild-type, programmed death-ligand 1
(PD-L1).
[0057] The technology described in the embodiments above are
intended to be illustrative examples. Variations and extensions of
these embodiments are envisioned as within the scope of the
invention. For example, multiple capture antibodies may be used to
enable comprehensive enrichment of an entire population of
heterogeneous CTCs. Although the embodiments are described using
EpCAM enrichment, the nanowell assay is highly sensitive and
specific to tumor-derived cells neglected by immunocytochemistry.
Moreover, the magnetic sifter can be generalized to accommodate
multiple cell capture antibodies instead of, or in conjunction
with, EpCAM. This would provide detection of other cancers that
shed CTCs (e.g. human epidermal growth factor receptor (HER2) for
breast cancer, neuron-glial antigen 2 (NG2) for melanoma, and
carbonic anhydrase IX (CAIX) for renal cell carcinoma) and to
EpCAM-low and EpCAM-negative populations. In addition, the
invention is not limited to 4 fluorophores for multiplex
single-cell gene expression profiling. As additional primer-probe
sets become commercially available, additional or alternative ones
may be used as well. Moreover, further modifications, including
laser excitation, sharper band-pass filters, and narrower-emission
hydrolysis probes, may extend the platform's multiplex capability
to accommodate 12 or more genes. Such expansion allows
comprehensive mutational profiling to develop along with clinical
advances to capture a comprehensive panel of relevant "actionable"
mutations for therapy selection and disease monitoring.
[0058] In conclusion, embodiments of the invention provide a
massively parallel, multigene profiling nano-platform and method to
analyze hundreds of single CTCs. It features a magnetic sifter for
high-efficiency CTC enrichment from blood and a single-cell
nanowell array for CTC mutation profiling using modular gene
panels. This approach to interrogate individual CTCs has
unprecedented sensitivity. To our knowledge, this is the first
demonstration of a high-throughput, multiplexed strategy for
single-cell gene mutation profiling of CTCs to provide minimally
invasive cancer therapy prediction and disease monitoring.
* * * * *