U.S. patent application number 15/222828 was filed with the patent office on 2016-11-17 for reaction mixtures for detecting nucleic acids altered by cancer in peripheral blood.
The applicant listed for this patent is VIOMICS Inc.. Invention is credited to David Mallery, Scott Morris.
Application Number | 20160333424 15/222828 |
Document ID | / |
Family ID | 48745455 |
Filed Date | 2016-11-17 |
United States Patent
Application |
20160333424 |
Kind Code |
A1 |
Morris; Scott ; et
al. |
November 17, 2016 |
REACTION MIXTURES FOR DETECTING NUCLEIC ACIDS ALTERED BY CANCER IN
PERIPHERAL BLOOD
Abstract
Analyzing peripheral blood RNA populations presents an
effective, accurate, minimally invasive method of determining a
patient's cancer status. Using circulating free RNA of the genes
disclosed herein, systems and methods are disclosed which can
accurately identify cancer signatures in the patient blood
samples.
Inventors: |
Morris; Scott; (Phoenix,
AZ) ; Mallery; David; (Solana Beach, CA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
VIOMICS Inc. |
Phoenix |
AZ |
US |
|
|
Family ID: |
48745455 |
Appl. No.: |
15/222828 |
Filed: |
July 28, 2016 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
13734735 |
Jan 4, 2013 |
9422592 |
|
|
15222828 |
|
|
|
|
61584097 |
Jan 6, 2012 |
|
|
|
Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C12Q 1/68 20130101; C12Q
2600/158 20130101; C12Q 1/6886 20130101 |
International
Class: |
C12Q 1/68 20060101
C12Q001/68 |
Claims
1. A reaction mixture comprising: a biological sample suspected of
containing circulating free nucleic acids derived from a target
formylpeptide receptor gene; one or more oligonucleotide probes
specific for the circulating free nucleic; and amplification
reagents for amplifying the circulating free nucleic acids using
the one or more oligonucleotide probes.
2. The reaction mixture of claim 1, wherein the target
formylpeptide receptor gene is FPR1.
3. The reaction mixture of claim 1, wherein the biological sample
comprises blood components.
4. The reaction mixture of claim 1, wherein the circulating free
nucleic acids comprises circulating free RNA transcripts derived
from the target formylpeptide receptor gene.
5. The reaction mixture of claim 1, wherein the one or more
oligonucleotide probes comprise an amplification primer, a
sequencing primer, a microarray probe, or a combination of any
thereof.
6. The reaction mixture of claim 1, wherein at least one of the one
or more oligonucleotide probes comprises a nucleotide sequence of
SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3.
7. The reaction mixture of claim 1, wherein the amplification
reagents comprise one or more of the following reagents: nucleic
acid polymerases, reverse transcriptases, DNAses, RNases, reaction
buffers, reducing agents, surfactants, divalent cations, and
dNTPs.
8. The reaction mixture of claim 1, wherein the reaction mixture is
a qPCR mixture reaction or an RT-PCR mixture reaction.
9. A kit for determining cancer risk in a subject, comprising one
or more oligonucleotide probes specific for circulating free
nucleic acids derived from a formylpeptide receptor gene and a
quantification standard for quantifying the level of circulating
free formylpeptide receptor nucleic acids in, the subject.
10. The kit of claim 9, wherein the quantification standard
comprises synthetic formylpeptide receptor nucleic acids.
11. The kit of claim 9, wherein the quantification standard
comprises at least three synthetic nucleic acid molecules having
the nucleotide sequences set forth in SEQ ID NO: 10, SEQ ID NO: 11,
and SEQ ID NO: 12, respectively.
12. The kit of claim 9, further comprising oligonucleotide probes
specific for circulating free nucleic acids derived from one or
more reference genes.
13. The kit of claim 12, wherein the one or more reference genes
comprise an Actin B gene (ACTB) or a heterogeneous nuclear
ribonucleoprotein gene (HNRNPA1).
14. The kit of claim 13, wherein at least one of the one or more
probes specific for circulating free nucleic acids derived from
ACTB comprises a nucleotide sequence of SEQ ID NO: 4, SEQ ID NO: 5,
or SEQ ID NO: 6.
15. The kit of claim 13, wherein at least one of the one or more
probes specific for circulating free nucleic acids derived from
HNRNPA1 comprises a nucleotide sequence of SEQ ID NO: 7, SEQ ID NO:
8, or SEQ ID NO: 9.
16. The kit of claim 8, wherein, the kit is configured to determine
lung cancer risk.
17. The kit of claim 16, wherein the kit is configured to determine
non-small cell lung cancer risk.
18. The kit of claim 8, further comprising reagents suitable for
the lysis of cells.
19. The kit of claim 9, further comprising reagents for the
isolation of circulating free nucleic acids, and/or for the storage
of the circulating free nucleic acids.
20. The kit of claim 9, further comprising a notice, in the form
prescribed by a governmental agency regulating the manufacture,
sale, or use of pharmaceutical or biological products, which
reflects approval by the agency of manufacture, sale, or use for
human administration.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation, application of U.S.
patent application Ser. No. 13/734,735, filed on Jan. 4, 2013,
entitled "System and Method of Detecting RNAs Altered by Cancer in
Peripheral Blood", which claims priority to U.S. Provisional Patent
Application 61/584,097, filed on Jan. 6, 2012 and entitled "System
and Method of Detecting RNAs Altered by Cancer in Peripheral Blood"
The disclosures of each of the above-described applications are
hereby incorporated herein by reference in their entireties.
SEQUENCE LISTING
[0002] This application is being filed along with a sequence
listing in Electronic format. The Sequence Listing is provided as a
file entitled VIOMC_003C1_SEQUENCE_LISTING.TXT, created Jul. 19,
2016, which is approximately 3 kb in size. The information in the
electronic format of the sequence listing is incorporated herein by
reference in its entirety in its entirety.
FIELD OF THE INVENTION
[0003] Systems and methods for detecting cancer by assaying
extracts from patient blood are provided. In particular, methods
for detecting circulating free RNA (cfRNA) levels and relationships
that are highly specific to patients with certain cancers are
provided.
BACKGROUND
[0004] Cancer is a major health risk in the United States and
internationally. Treatments exist, but are often not administered
to patients until the disease has progressed to a point at which
treatment efficacy is compromised.
[0005] A major challenge in, cancer treatment is to identify
patients early in the course of their disease. This is difficult
under current methods because early cancerous or precancerous cell
populations may be asymptomatic and may be located in regions which
are difficult to access by biopsy. Thus a robust, minimally
invasive assay that may be used to identify all stages of the
disease, including early stages which may be asymptomatic, would be
of substantial benefit for the treatment of cancer.
SUMMARY OF THE INVENTION
[0006] The systems, devices, kits, and methods disclosed herein
each have several aspects, no single one of which is solely
responsible for their desirable attributes. Without limiting the
scope of the claims, some prominent features will now be discussed
briefly. Numerous other embodiments are also contemplated,
including embodiments that have fewer, additional, and/or different
components, steps, features, objects, benefits, and advantages. The
components, aspects, and steps may also be arranged and ordered
differently. After considering this discussion, and particularly
after reading the section entitled "Detailed Description," one will
understand how the features of the devices and methods disclosed
herein provide advantages over other known devices and methods.
[0007] One embodiment is a method for detection of one or more RNA
molecules in samples taken from a patient. In this embodiment,
blood or blood component such as plasma is isolated from a patient
suspected of having lung cancer or non-small cell lung cancer
(NSCLC). The plasma is analyzed to measure the level of circulating
free RNA (cfRNA) from one or more genes. In some embodiments the
RNA to be measured is messenger ribonucleic acids (mRNA), such as
mRNA from within the population of cfRNA in a patient's plasma. In
some embodiments the level of circulating free actin beta (ACTB)
RNA is measured. In some embodiments the level of circulating free
HNRNPA1 RNA is measured. In some embodiments, the level of
circulating free formylpeptide receptor gene (FPR1) RNA is
measured. In some embodiments, the level of cfRNA from FPR1, as
compared with the level of cfRNA from ACTB and HNRNPA1 is compared,
as discussed below, to determine if a patient is at risk for having
lung cancer or NSCLC. In some embodiments the NSCLC assayed is
Stage I NSCLC.
[0008] In some embodiments, at least one subset of one or more RNAs
(subset #2) is known to be present in plasma of cancer-free
individuals in relatively consistent quantities, and is known to be
present at a level different from this generally consistent level
in individuals with cancer such as breast, colon or lung cancer.
For example, levels may be consistently lower in, for example
patients having non-small cell lung cancer (NSCLC), such as stage I
NSCLC. In some embodiments levels may be consistently higher in
patients having cancer, for example patients having non-small cell
lung cancer (NSCLC), such as stage I NSCLC. In some embodiments the
lung cancer of said individuals does not show metastasis to the
lymph nodes. In some embodiments, the RNA(s) selected for subset #1
may be present in, lower levels due to nuclease activity, and in
some embodiments the RNA or region of the RNA assayed for may be
highly sensitive to nucleases due to the presence of nuclease
cleavage sites or the presence of secondary structures (i.e. double
vs. single stranded) that are preferentially cleaved by RNases. In
some embodiments the RNA selected for subset #1 may be represented
in higher levels in certain cancer patients, such as lung cancer
patients. In some embodiments, the change in levels of RNA
accumulation in subset #1 may be due to effects of molecules
secreted by cancers or immune cells within close proximity to
tumors on other non-malignant tissues.
[0009] In some embodiment, RNAs in subset #1 are released into the
blood at increased levels in cancer patients due to one or more of
the following: 1) preferential release from cells into the blood,
2) increased abundance yin tumor cells, 3) increased abundance in
cells near the tumor (i.e. reactive cells or stroma), 4) increased
abundance from cells not near the tumor, mediated by secreted
signals.
[0010] Another subset of one or more RNAs (subset #2) may be known
to be stably expressed in all individuals regardless of cancer
status. In some embodiments, the RNAs in subset #2 are less
sensitive to ribonucleases.
[0011] In some embodiments, relative abundance of markers of subset
#1 and subset #2 may be indicative of the presence of cancerous
cells, tumors, or cells with a heightened potential to become
cancerous, and in other embodiments, the accumulation levels of
markers in subset #1 alone may be indicative of cancerous cells,
tumors, or cells with a heightened potential to become cancerous.
In some embodiments the RNA levels of a subset #2 member or members
may vary inversely with the levels of a subset #1 member or member,
such that the ratio of the levels is indicative of cancer
status.
[0012] In some embodiments, digital PCR or real-time PCR is used as
a detection method to determine the presence or accumulation levels
of RNA markers. In some embodiments, DNA sequencing may be used as
a detection method to determine the quantity of RNA markers. In
other embodiments, detection may occur by molecular barcoding
technologies such as NanoString or nCounter.
[0013] In some embodiments, the results of RNA and/or DNA copy
number are analyzed to determine an assay outcome (i.e., positive
or negative result) based at least in part on statistical distances
between results. In some aspects, patients may be classified into
different risk groups based at least in part on the analysis of the
relative abundance of markers as disclosed herein. The cumulative
distribution function of the normal, binomial and/or Poisson
distribution or similar functions may be used to determine relative
abundance of RNAs. In some embodiments, the type of cancer present
in a patient may be predicted at least in part on results of RNA
expression.
BRIEF DESCRIPTION OF FIGURE DRAWINGS
[0014] FIGS. 1A, 1B, and 1C depicts box plots of accumulation
levels for the markers FPR1, ACTB and HNRNPA1, respectively, from
plasma taken from normal individuals and individuals known to have
one or more tumors. The y-axis indicates the transcript
accumulation level per mL of plasma assayed, normalized against
accumulation levels measured in Universal Human Reference RNA,
represented natural logarithmically. According to the standard
convention for boxplots, the central horizontal line in each column
represents the median, the box represents the 25%-75% quartiles and
the error bars indicate the extreme observations (excluding
outliers). All units are ratios of transcript accumulation levels
per mL of plasma assayed relative to accumulation levels of similar
transcripts obtained from 1 ng of Agilent's Universal Human
RNA.
[0015] FIG. 2A depicts two-dimensional scatter-plots of ratios ACTB
and HNRNPA1 values normalized to FPR1 values, wherein sample values
are normalized to values determined from RNA obtained from a cell
line control. Dashed line A separates the majority of values
obtained from Normal patients. Dashed, line B separates the
majority of values obtained from NSCLC patients. The values between
dashed lines A and B represent the minimal degree of overlap
between the highest Normal values and the lowest NSCLC values.
[0016] FIG. 2B depicts two-dimensional scatter-plots of ratios ACTB
and HNRNPA1 values normalized to FPR1 values, wherein sample values
are normalized to values determined from RNA obtained from a
synthetic standard. The dashed diagonal line passing through the
points (0.1, 10), (1, 1) and (10, 0.1) separates a population of
normal samples (white-filled circles) from a population of
predominantly NSCLC samples (black-filled circles).
DETAILED DESCRIPTION
[0017] One embodiment relates systems and methods for determining
whether a patient at risk for cancer may have the disease by
analyzing circulating nucleic acids in the blood. Determination of
patients that may have cancer may be done on blood-derived
specimens to assay RNA accumulation levels, and such analysis may
be conducted by expression microarray, sequencing, nCounter, or
real-time PCR. In some embodiments, expression levels of first
subset of control nucleic acids are compared to expression levels
of a second subset of nucleic acids that are known to be increased
in patients having cancer. The first subset of control nucleic
acids may be found by analyzing plasma from many disease-free
patients and selecting genes that are expressed at stable levels
within those patients. Subsets may also be found by analyzing solid
tissue specimens taken from multiple tissue types (i.e. colon,
lung, kidney, liver, etc.), and selecting genes that are expressed
as circulating free nucleic acids at stable levels in a patient's
blood.
[0018] In some embodiments, Subset #1 can be selected by analyzing
genes whose transcript accumulation levels increase in plasma or in
solid tumor specimens.
[0019] In some embodiments, Subset #1 includes genes whose
circulating free nucleic acid levels decrease in plasma or yin
solid tumor specimens taken from individuals suffering from
cancer.
[0020] In some embodiments, subset #1 comprises genes whose
transcript accumulation levels are unchanged in normal individuals
as compared to cancer patients. In these embodiments subset #2 is
selected, in combination with one or more genes of subset #1 whose
accumulation levels increase in plasma or in solid tumors
specimens.
[0021] In some embodiment, aspects of the invention relate to the
discovery that circulating free RNA (cfRNA) levels of formylpeptide
receptor gene (FPR1) RNA change in patients suffering from cancer.
For example, cfRNA levels of FPR1 were found to increase in
patients having lung cancer, as described below. Moreover, cfRNA
levels of FPR1 were shown to increase in comparison to cfRNA levels
of other genes, such as ACTB and HNRNPA1 or other transcripts
listed in subset #2.
[0022] As shown in FIG. 1A and described with reference to Example
1, FIG. 1A depicts transcript accumulation levels for the gene FPR1
in samples measured from plasma taken from patients classified as
having a cancer status as either normal (i.e., putatively cancer
free) and tumor cells (i.e., having known tumor cells). The y-axis
logarithmic values indicate that the FPR1 transcript accumulates on
average at about a 100-fold greater level in tumor cell patients as
compared to normal patients.
[0023] FIG. 1B and FIG. 1C depict transcript accumulation levels
for the genes ACTB and HNRNPA1, respectively, in samples measured
from plasma taken from patients classified as having a cancer
status as either normal (i.e., putatively cancer free) and tumor
cells (i.e., having known tumor cells). The y-axis logarithmic
values indicate that the ACTB and HNRNPA1 transcripts accumulate on
average at levels which are comparable in normal and in tumor cell
patients.
[0024] In some embodiments, once subset #1 is known, subset #2 can
be selected by analyzing a large number of candidates from multiple
specimens and selecting those for which the difference between
subset #2 and subset #1 is largest in plasma from cancer patients.
In some embodiments, subset #2 can be selected by surveying
transcript accumulation levels of many genes and finding which ones
have the lowest variability. In some embodiments genes are selected
not based on their individual accumulation levels but on the lack
of change in their relative accumulation levels in cancer.
[0025] FIGS. 1A-C indicate that FPR1 and ACTB, FPR1 and HNRNPA1, or
FPR1 and both ACTB and HNRNPA1 are suitable combinations of
subset#1 and subset #2 genes for the methods disclosed herein,
although embodiments of the invention are not limited to only these
genes.
[0026] Once subset #1 (and subset #2 in some embodiments) are known
within a given cancer type, the expression profile can be measured
in plasma taken from cancer patients and patients for which a
cancer is to be assayed. Because plasma can be collected and
prepared within many primary care physician offices without posing
any more risk than a standard blood draw, relative cfRNA
accumulation levels between subsets #1 and subset #2 in some
embodiments may be a valuable cancer biomarker. Additionally, if
subsets #1 and subset #2 in some embodiments may be assayed
reliably, they may have a number of advantages over current cancer
assays. For example, in some embodiments this method may detect
cancer at an early stage of development, cancer that poses few
symptoms, cancer that is difficult to distinguish from benign
conditions or cancer that may be developing in an area of the body
that may not be accessible to traditional biopsy assays.
[0027] Increased RNase activity is often present in tumors. This
RNase activity may inhibit tumor growth, and may be part of the
immune system's response to cancer. Cytotoxic T cells may lead to
apoptosis of cancer cells via IFN-.gamma., and this apoptosis may
result in activation of RNases, such as RNase L. Death of cells via
necrosis, which may be caused by hypoxia due to tumor growth, may
also contribute to the release of RNases. It is known that plasma
of lung cancer patients has increased RNase activity (Marabella et
al., (1976) "Serum ribonuclease in patients with lung carcinoma,"
Journal of Surgical Oncology, 8(6):501-505; Reddi et al. (1976)
"Elevated serum ribonuclease in patients with pancreatic cancer,"
Proc. Nat'l. Acad. Sci. USA 73(7):2308-2310). It is also known that
lung cells contain RNases similar to those found in plasma (Neuwelt
et al., (1978) "Possible Sites of Origin of Human Plasma
Ribonucleases as Evidenced by Isolation and Partial
Characterization of Ribonucleases from Several Human Tissues,"
Cancer Research 38:88-93).
[0028] When higher levels of RNase are present in plasma, any free
RNA is susceptible to more rapid degradation. Thus, there may be
less RNA detectable in plasma RNA preparations. While all RNA may
be present at decreased levels, it is only possible to detect this
difference with any level of accuracy when the normal variability
of a gene is low. For example, if the normal range of a gene's
expression is between 10 and 100 units, it may be difficult to
accurately detect a decrease of 1 unit. However, if a gene's
expression is normally between 10 and 11 units, a decrease of 1
unit is readily detectable (i.e. any number under 10 units would
indicate a decrease).
[0029] FPR1 plays multiple roles in the lungs and cancer. FPR1 is
expressed in lung fibroblasts (VanCompernolle et al. (2003) J
Immunol. 171(4):2050-6) and is necessary for wound repair in the
lungs (Shao (2011) Am J Respir Cell Mol Biol 44:264-269). It is
known that fibroblasts are important in both attracting immune
cells that fight the tumor (Gemperle (2012) PLOSOne 7(11):1-7,
e50195) and creation of stroma which protects the tumor (Wang
(2009) Clin Cancer Res 15(21) 6630-6638). FPR1 may also exacerbate
the activity of other oncogenes in tumors (Huang (2007) Cancer Res
67(12):5906-5913). There is no evidence that it is overexpressed in
lung cancers, but FPR1 is known to be regulated by RNA
stabilization (Mandal (2007) J Immunol 178:2542-2548, Mandal (2005)
J Immunol 175:6085-6091). Given these roles, it is possible that
FPR1 RNA is secreted deliberately by either tumor cells to enhance
tumor growth (i.e. by activating wound-repair systems for growth or
growing protective stroma) or immune cells to enhance the immune
response (i.e. attracting additional immune cells).
[0030] In some embodiments the method can begin by extracting cfRNA
from a patient's sample and assaying the cfRNA extracted. See,
e.g., O'Driscoll, L. et al. (2008) "Feasibility and relevance of
global expression profiling of gene transcripts in serum from
breast cancer patients using whole genome microarrays and
quantitative RT-PCR." Cancer Genomics Proteomics 5:94-104, which is
hereby incorporated by reference in its entirety. In some
embodiments, a consistent, repeatable method is used to isolate
cfRNA from plasma or other source of RNA to ensure the reliability
of the data. To obtain cfRNA from blood, one may use the protocol
listed below although other methods are also contemplated.
[0031] cfRNA molecules may be purified from plasma or other samples
using, for example, Qiagen's QIAamp circulating nucleic acid kit.
The protocol in this kit is described in the document "QIAamp
Circulating Nucleic Acid Handbook", Second Edition, January 2011,
which is hereby incorporated, by reference in its entirety. This
protocol provides an embodiment of a method to purify circulating
total nucleic acid from 1 mL of plasma. In brief, lysis reagents
and proteases are added along with inert carrier RNA. The total
nucleic acid (DNA and RNA) is bound to a column, and the column is
washed multiple times then eluted off the column.
[0032] For example the protocol may be performed by executing the
steps as follows. Pipet 100 .mu.l, 200 .mu.l, or 300 .mu.l QIAGEN
Proteinase K into a 50 ml centrifuge tube. Add 1 ml, 2 ml, or 3 ml
of serum or plasma to the 50 ml tube. Add 0.8 ml, 1.6 ml, or 2.4 ml
Buffer ACL (containing 1.0 .mu.g carrier RNA). Close the cap and
mix by pulse-vortexing for 30 s, making sure that a visible vortex
forms in, the tube. In order to ensure efficient lysis, mix the
sample and Buffer ACL thoroughly to yield a homogeneous solution.
The procedure should not be interrupted at this time.
[0033] To start the lysis incubation, incubate at 60.degree. C. for
30 min. Place the tube back on the lab bench and add 1.8 ml, 3.6
ml, or 5.4 ml Buffer ACB to the lysate in the tube. Close the cap
and mix thoroughly by pulse-vortexing for 15-30 seconds. Incubate
the lysate-Buffer ACB mixture in the tube for 5 min on ice. Insert
the QIAamp Mini column into the VacConnector on the QIAvac 24 Plus.
Insert a 20 ml tube extender into the open QIAamp Mini column. Make
sure that the tube extender is firmly inserted into the QIAamp Mini
column in order to avoid leakage of sample.
[0034] Keep the collection tube for the dry spin, below. Carefully
apply the lysate-Buffer ACB mixture into the tube extender of the
QIAamp Mini column. Switch on the vacuum pump. When all lysates
have been drawn through the columns completely, switch off the
vacuum pump and release the pressure to 0 mbar. Carefully remove
and discard the tube extender. Please note that large sample lysate
volumes (about 11 ml when starting with 3 ml sample) may need up to
10 minutes to pass through the QIAamp Mini membrane by vacuum
force. For fast and convenient release of the vacuum pressure, the
Vacuum Regulator should be used (part of the QIAvac Connecting
System). To avoid cross-contamination, be careful not to move the
tube extenders over neighboring QIAamp Mini Columns.
[0035] Apply 600 .mu.l Buffer ACW1 to the QIAamp Mini column. Leave
the lid of the column open, and switch on the vacuum pump. After
all of Buffer ACW1 has been drawn through the QIAamp Mini column,
switch off the vacuum pump and release the pressure to 0 mbar.
Apply 750 .mu.l Buffer ACW2 to the QIAamp Mini column. Leave the
lid of the column open, and switch on the vacuum pump. After all of
Buffer ACW2 has been drawn through the QIAamp Mini column, switch
off the vacuum pump and release the pressure to 0 mbar. Apply 750
.mu.l of ethanol (96-100%) to the QIAamp Mini column. Leave the lid
of the column open, and switch on the vacuum pump. After all of
ethanol has been drawn through the spin column, switch off the
vacuum pump and release the pressure to 0 mbar. Close the lid of
the QIAamp Mini column. Remove it from the vacuum manifold, and
discard the VacConnector. Place the QIAamp Mini column, in a clean
2 ml collection tube, and centrifuge at full, speed
(20,000.times.g; 14,000 rpm) for 3 min.
[0036] Place the QIAamp Mini Column into a new 2 ml collection
tube. Open the lid, and incubate the assembly at 56.degree. C. for
10 min to dry the membrane completely. Place the QIAamp Mini column
in a clean 1.5 ml elution tube (provided) and discard the 2 ml
collection tube from step 14. Carefully apply 20-150 .mu.l of
Buffer AVE to the center of the QIAamp Mini membrane. Close the lid
and incubate at room temperature for 3 min. Ensure that the elution
buffer AVE is equilibrated to room temperature (15-25.degree. C.).
If elution is done in small volumes (<50 .mu.l) the elution
buffer has to be dispensed onto the center of the membrane for
complete elution of bound DNA. Elution volume is flexible and can
be adapted according to the requirements of downstream
applications. The recovered eluate volume will be up to 5 .mu.l
less than the elution volume applied to the QIAamp Mini column.
Centrifuge in a microcentrifuge at full speed (20,000.times.g;
14,000 rpm) for 1 min to elute the nucleic acids. The above example
QIAamp Circulating Nucleic Acid Handbook 1/2011 is representative
on, knowledge of one of skill in the art and it, illustrative
rather than limiting. Alternate embodiments, including variants on
the methods above or distinct approaches to cfRNA purification, are
contemplated herein, and the methods and compositions disclosed
herein are not limited to any particular cfRNA purification
method.
[0037] Samples produced by this method may be highly pure and free
of PCR inhibitors, and may be suitable for qPCR as used in some
embodiments to assay cfRNA relative expression as an assay of, for
example, various types of cancer.
[0038] In some embodiments the methods include performing PCR or
qPCR in order to generate an amplicon. Numerous different PCR and
qPCR protocols are known in the art and exemplified herein below
and can be directly applied or adapted for use using the presently
described compositions for the detection and/or identification
of
[0039] Some embodiments provide methods including Quantitative PCR
(qPCR) (also referred as real-time PCR). qPCR can provide
quantitative measurements, and also provide the benefits of reduced
time and contamination. As used herein, "quantitative PCR" ("qPCR"
or more specifically "real time qPCR") refers to the direct
monitoring of the progress of a PCR amplification as it is
occurring without the need for repeated sampling of the reaction
products. In qPCR, the reaction products may be monitored via a
signaling mechanism (e.g., fluorescence) as they are generated and
are tracked after the signal rises above a background level but
before the reaction, reaches a plateau. The number of cycles
required to achieve a detectable or "threshold" level of
fluorescence (herein referred to as cycle threshold or "CT") varies
directly with the concentration of amplifiable targets at the
beginning of the PCR process, enabling a measure of signal
intensity to provide a measure of the amount of target nucleic acid
in a sample in real time.
[0040] Methods for setting up PCR and qPCR are well known to those
skilled in the art. The reaction mixture minimally comprises
template nucleic acid (e.g., as present in test samples, except in
the case of a negative control as described below) and
oligonucleotide primers and/or probes in combination with suitable
buffers, salts, and the like, and an appropriate concentration of a
nucleic acid polymerase. As used herein, "nucleic acid polymerase"
refers to an enzyme that catalyzes the polymerization of nucleoside
triphosphates. Generally, the enzyme will initiate synthesis at the
3'-end of the primer annealed to the target sequence, and will
proceed in the 5'-3' direction along the template until synthesis
terminates. An appropriate concentration includes one that
catalyzes this reaction in the presently described methods. Known
DNA polymerases useful in the methods disclosed herein include, for
example, E. coli DNA polymerase I, T7 DNA polymerase, Thermus
thermophilus (Tth) DNA polymerase, Bacillus stearothermophilus DNA
polymerase, Thermococcus litoralis DNA polymerase, Thermus
aquaticus (Taq) DNA polymerase and Pyrococcus furiosus (Pfu) DNA
polymerase, FASTSTART.TM. Taq DNA polymerase, APTATAQ.TM. DNA
polymerase (Roche), KLENTAQ 1.TM. DNA polymerase (AB peptides
Inc.), HOTGOLDSTAR.TM. DNA polymerase (Eurogentec), KAPATAQ.TM.
HotStart DNA polymerase, KAPA2G.TM. Fast HotStart DNA polymerase
(Kapa Biosystemss), PHUSION.TM. Hot Start DNA Polymerase
(Finnzymes), or the like.
[0041] In addition to the above components, the reaction mixture of
the present methods includes primers, probes, and
deoxyribonucleoside triphosphates (dNTPs).
[0042] Usually the reaction mixture will further comprise four
different types of dNTPs corresponding to the four naturally
occurring nucleoside bases, i.e., dATP, dTTP, dCTP, and dGTP. In
some embodiments, each dNTP will typically be present in an amount
ranging from about 10 to 5000 .mu.M, usually from about 20 to 1000
.mu.M, about 100 to 800 .mu.M, or about 300 to 600 .mu.M.
[0043] The reaction mixture can further include an aqueous buffer
medium that includes a source of monovalent ions, a source of
divalent cations, and a buffering agent. Any convenient source of
monovalent ions, such as potassium chloride, potassium acetate,
ammonium acetate, potassium glutamate, ammonium chloride, ammonium
sulfate, and the like may be employed. The divalent cation may be
magnesium, manganese, zinc, and the like, where the cation will
typically be magnesium. Any convenient source of magnesium cation
may be employed, including magnesium chloride, magnesium acetate,
and the like. The amount of magnesium present in the buffer may
range from 0.5 to 10 mM, and can range from about 1 to about 6 mM,
or about 3 to about 5 mM. Representative buffering agents or salts
that may be present in the buffer include Tris, Tricine, HEPES,
MOPS, and the like, where the amount of buffering agent will
typically range from about 5 to 150 mM, usually from about 10 to
100 mM, and more usually from about 20 to 50 mM, where in certain
preferred embodiments the buffering agent will be present in an
amount sufficient to provide a pH ranging from about 6.0 to 9.5,
for example, about pH 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, or 9.5.
Other agents that may be present in the buffer medium include
chelating agents, such, as EDTA, EGTA, and the like. In some
embodiments, the reaction mixture can include BSA, or the like. In
addition, in some embodiments, the reactions can include a
cryoprotectant, such as trehalose, particularly when the reagents
are provided as a master mix, which can be stored over time.
[0044] In preparing a reaction mixture, the various constituent
components may be combined in any convenient order. For example,
the buffer may be combined with primer, polymerase, and then
template nucleic acid, or all of the various constituent components
may be combined at the same time to produce the reaction
mixture.
[0045] Alternatively, commercially available premixed reagents can
be utilized in the methods disclosed herein, according to the
manufacturer's instructions, or modified to improve reaction
conditions (e.g., modification of buffer concentration, cation
concentration, or dNTP concentration, as necessary), including, for
example, Quantifast PCR mixes (Qiagen), TAQMAN.RTM. Universal PCR
Master Mix (Applied Biosystems), OMNIMIX.RTM. or SMARTMIX.RTM.
(Cepheid), IQ™ Supermix (Bio-Rad Laboratories), LIGHTCYCLER.RTM.
FastStart (Roche Applied Science, Indianapolis, Ind.), or
BRILLIANT.RTM. QPCR Master Mix (Stratagene, La Jolla, Calif.).
[0046] The reaction mixture can be subjected to primer extension
reaction conditions ("conditions sufficient to provide
polymerase-based nucleic acid amplification products"), i.e.,
conditions that permit for polymerase-mediated primer extension by
addition of nucleotides to the end of the primer molecule using the
template strand as a template. In many embodiments, the primer
extension reaction conditions are amplification conditions, which
conditions include a plurality of reaction cycles, where each
reaction cycle comprises: (1) a denaturation step, (2) an annealing
step, and (3) a polymerization step. As discussed below, in some
embodiments, the amplification protocol does not include a specific
time dedicated to annealing, and instead comprises only specific
times dedicated to denaturation and extension. The number of
reaction cycles will vary depending on the application being
performed, but will usually be at least 15, more usually at least
20, and may be as high as 60 or higher, where the number of
different cycles will typically range from about 20 to 40. For
methods where more than about 25, usually more than about 30 cycles
are performed, it may be convenient or desirable to introduce
additional polymerase into the reaction mixture such that
conditions suitable for enzymatic primer extension are
maintained.
[0047] The denaturation step comprises heating the reaction mixture
to an elevated temperature and maintaining the mixture at the
elevated temperature for a period of time sufficient for any
double-stranded or hybridized nucleic acid present in the reaction
mixture to dissociate. For denaturation, the temperature of the
reaction mixture will usually be raised to, and maintained, at, a
temperature ranging from about 85 to 100.degree. C., usually from
about 90 to 98.degree. C., and more usually from about 93 to
96.degree. C., for a period of time ranging from about 3 to 120
sec, usually from about 3 sec.
[0048] Following denaturation, the reaction mixture can be
subjected to conditions sufficient for primer annealing to template
nucleic acid present in, the mixture (if present), and for
polymerization of nucleotides to the primer ends in a manner such
that the primer is extended in a 5' to 3' direction using the
nucleic acid to which it is hybridized as a template, i.e.,
conditions sufficient for enzymatic production of primer extension
product. In some embodiments, the annealing and extension processes
occur in the same step. The temperature to which the reaction
mixture is lowered to achieve these conditions will usually be
chosen to provide optimal efficiency and specificity, and will
generally range from about 50 to 85.degree. C., usually from about
55 to 70.degree. C., and more usually from about 60 to 68.degree.
C. In some embodiments, the annealing conditions can be maintained
for a period of time ranging from about 15 sec to 30 min, usually
from about 20 sec to 5 min, or about 30 sec to 1 minute, or about
30 seconds.
[0049] This step can optionally comprise one of each of an
annealing step and an extension step with variation and
optimization of the temperature and length of time for each step.
In a two-step annealing and extension, the annealing step is
allowed to proceed as above. Following annealing of primer to
template nucleic acid, the reaction mixture will be further
subjected to conditions sufficient to provide for polymerization of
nucleotides to the primer ends as above. To achieve polymerization
conditions, the temperature of the reaction mixture will typically
be raised to or maintained at a temperature ranging from about 65
to 75.degree. C., usually from about 67 to 73.degree. C. and
maintained for a period of time ranging from about 15 sec to 20
min, usually from about 30 sec to 5 min. In some embodiments, the
methods disclosed herein do not include a separate annealing and
extension step. Rather, the methods include denaturation and
extension steps, without any step dedicated specifically to
annealing.
[0050] The above cycles of denaturation, annealing, and extension
may be performed using an automated device, typically known as a
thermal cycler. Thermal cyclers that may be employed are described
elsewhere herein as well as in U.S. Pat. Nos. 5,612,473; 5,602,756;
5,538,871; and 5,475,610; the disclosures of which are herein
incorporated by reference.
[0051] The methods described herein can also be used in non-PCR
based applications to detect a target nucleic acid sequence, where
such target may be immobilized on a solid support. Methods of
immobilizing a nucleic acid sequence on a solid support are known
in the art and are described in Ausubel et al, eds. (1995) Current
Protocols in Molecular Biology (Greene Publishing and
Wiley-Interscience, NY), and in protocols provided by the
manufacturers, e.g., for membranes: Pall Corporation, Schleicher
& Schuell; for magnetic beads: Dynal; for culture plates:
Costar, Nalgenunc; for bead array platforms: Luminex and Becton
Dickinson; and, for other supports useful according to the
embodiments provided herein, CPG, Inc.
[0052] Variations on the exact amounts of the various reagents and
on the conditions for the PCR or other suitable amplification
procedure (e.g., buffer conditions, cycling times, etc.) that lead
to similar amplification or detection/quantification results are
known to those of skill in the art and are considered to be
equivalents. In one embodiment, the subject qPCR detection has a
sensitivity of detecting fewer than 50 copies (preferably fewer
than 25 copies, more preferably fewer than 15 copies, still more
preferably fewer than 10 copies, e.g. 5, 4, 3, 2, or 1 copy) of
target nucleic acid in a sample.
[0053] In some embodiments the method may involve PCR amplification
of cfRNA template RNA. A DNase treatment may be conducted to remove
DNA contamination from RNA samples. cfRNA may be converted to cDNA
with a reverse transcriptase and this step may use one or more of
the same primers used within a PCR reaction. Target cDNAs may be
amplified by, for example, a consistent, repeatable method to
amplify cDNA from plasma or other cDNA. In some embodiments, one or
more targets in cDNA may be amplified and quantified via Taqman
chemistry. This protocol may not be the only suitable protocol to
detect cfRNA quantity. However, it may be important to use a
consistent protocol, for cDNA synthesis and amplification, as
variations in protocol may have a large effect on the eventual
results.
[0054] In some embodiments the method may involve an assay for
non-small cell lung cancers (NSCLC). In some embodiments, an assay
may involve one or more of the following genes to comprise subset
#2: PLGLB2, GABARAP, HNRNPA1, NACA, EIF1, UBB, UBC, CD81, TMBIM6,
MYL12B, ACTB, HSP90B1, CLDN18, RAMP2, MFAP4, FABP4, MARCO, RGL1,
ZBTB16, C10orf116, GRK5, AGER, SCGB1A1, HBB, TCF21, GMFG, HYAL1,
TEK, GNG11, ADH1A, TGFBR3, INPP1, ADH1B; and one or more of the
following genes to comprise subset #1: CTSS, FPR1, FPR2, FPRL1,
FPRL2, CXCR2, NCF2.
[0055] A proprietary R1b assay may be used. In this embodiment, the
assay may be a 3-plex qPCR assay that detects relative abundance of
ACTB, HNRNPA1 and FPR1. In some embodiments ACTB and HNRNPA1 may
fulfill the criteria for subset #2, and FPR1 may fulfill the
criteria for subset #1.
[0056] In some embodiments, the subset #2 may consist of ACTB and
subset #1 may consist of FPR1. In some embodiments the subset #2
may consist of HNRNPA1. In some embodiments the subset #2 may
consist of ACTB and HNRNPA1. In some embodiments the subset #2 may
comprise at least one of ACTB and HNRNPA1. In some embodiments
subset #1 is FPR1 and Subset #2 is ACTB, or HNRNP1, or both ACTB
and HNRNP1. In some embodiments, Qiagen assay #QF00119602 may be
used for the qPCR, using the primers/probes provided accorded to
the manufacturer's protocol. Agilent's Universal RNA may be used as
a standard in qPCR. In another embodiment, the R1b assay consisting
of the following primer/probes may be as follows in Table 1.
TABLE-US-00001 TABLE 1 Amplification Primers Gene Forward Primer
Probe Reverse Primer FPR1 TGACGGTGAGAGG [FAM] GGTGGCAATAAGCCCA
CATCA CGGTTCATCATTGGCTTCAG TAACTG (SEQ ID NO: 1) CGC [BHQ1] (SEQ ID
NO: 3) (SEQ ID NO: 2) ACTB AGGCCAACCGCGA [CAL Fluor Gold 540]
TGCCATCCTAAAAGCC GAAGA TGACCCAGATCATGTTTGAG ACCCCA (SEQ ID NO: 4)
ACCTTCA [BHQ1] (SEQ ID NO: 6) (SEQ ID NO: 5) HNRNPA1 GGGCTTTGCCTTTG
[Quasar 705] TGTGGCCATTCACAGT TAACCTT TGACGACCATGACTCCGTGG ATGGTA
(SEQ ID NO: 7) ATA [BHQ3] (SEQ ID NO: 9) (SEQ ID NO: 8)
[0057] An RNA standard may be used to standardize result across
multiple runs. This standard may be run at different dilutions. In
some embodiments a synthetic standard may be used. For example, the
normal ranges and cut-offs for one or more markers may be examined,
and synthetic standards may be obtained and used directly, or
diluted or combined such that they are at levels similar to
predicted levels, such as predicted levels of the markers. In some
embodiments the synthetic standards are present at levels that are
at or within an order of magnitude of (i.e., 10-fold higher or
10-fold lower than) predicted levels in a patient sample. In some
embodiments the synthetic standards are present at or within a
difference of 5.times. (either 5-fold higher or five-fold lower)
than levels predicted for a patient sample. In some embodiments the
synthetic standards are present at or within a difference of
2.times. (either 2-fold higher or 2-fold lower) than levels
predicted for a patient sample.
[0058] Many methods may be used to determine the appropriate level
of each synthetic RNA in the synthetic standard. In one embodiment,
one may run some number of samples representative of those and
record the results (i.e. Ct value or fitted value to a standard).
Each synthetic RNA may then be run on the same assay and the
results may be measured on the same scale as the samples (i.e. Ct
score or fitted value to a standard). Upon examination, one can
determine which standards must be used. For example, 50 samples may
be run and Ct scores ranging from 33-38 are obtained for a given
gene. Standards of 10.sup.7, 10.sup.6, 10.sup.5, 10.sup.4,
10.sup.3, 10.sup.2 copies per .mu.L may yield Ct scores of 24, 28,
32, 36, 40, or 44. Thus, it may be decided to use the 10.sup.5
standard, with dilutions to 10.sup.4 and 10.sup.3 conducted during
assay setup. Using this strategy, only the original standard and
two dilutions are needed to cover future samples. A similar method
could be used to select appropriate concentrations for other
standards in the same multiplex. Using this method, different
concentrations may be used for each transcript to be assayed so a
single standard can be used even if there are large discrepancies
between different genes in the multiplex. By using the method
disclosed herein, transcripts of widely ranging accumulation levels
may be assayed with a reduced number of amplification reactions on
standard templates.
[0059] For example, if one expects gene A to be in the range of 100
to 10,000 copies/.mu.l and gene B to be in the range of 1,000,000
to 100,000,000 copies, one may create a mixed synthetic standard of
10,000 copies gene A and 100,000,000 copies gene B, thereby only
requiring three standards in a 10-fold dilution series to cover the
whole range expected for a sample. Using such a synthetic standard
may in some embodiments dramatically reduce the number of standard
or control samples that need to be run in a qPCR reaction plate to
generate a standard curve that covers the expected ranges of both
gene a and gene B. This method will also minimize risk of small
errors introduced by pipetting from compounding during serial
dilutions.
[0060] Regression may be used to fit data points generated from
patient samples to the standard, such that results are expressed in
standard units. In some embodiments, the standard consists of RNA
created from one or more cell lines. In some embodiments, the
standard may consist of synthetic RNAs. The number of fragments of
each RNA within the standard may be known, and the standardized
unit may be number of RNA molecules present for each target. In
some embodiments, the standard may consist of the following
synthetic RNAs:
TABLE-US-00002 Target Synthetic RNA sequence FPR 1
5'CAUGUUGACGGUGAGAGGCAUCAUCCGGUUCAUCAUUG
GCUUCAGCGCACCCAUGUCCAUCGUUGCUGUCAGUUAUGG GCUUAUUGCCACCAAGAU3' (SEQ
ID NO: 10) ACTB 5'CCCCAAGGCCAACCGCGAGAAGAUGACCCAGAUCAUGU
UUGAGACCUUCAACACCCCAGCCAUGUACGUUGCUAUCCA GGCUGUGCUAUCCC3' (SEQ ID
NO: 11) HNRNPA1 5'AAAAGGGGCUUUGCCUUUGUAACCUUUGACGACCAUGA
CUCCGUGGAUAAGAUUGUCAUUCAGAAAUACCAUACUGUG AAUGGCCACAACUGU3' (SEQ ID
NO: 12)
[0061] Assays may involve components of different sequence or with
different detectable labels targeted to similar regions, components
targeted to different regions of the same genes, or components
targeting the regions of genes other than those listed in the R1a
assay above.
[0062] The results of an R1a test, may be evaluated using the
Decision Rules for Viomics' Test for cancer such, as Viomics' NSCLC
Test. A plot may be created where one axis is the ratio of FPR1 to
ACTB, and the other axis is the ratio of FPR1 to HNRNPA1. An
example of such a plot is indicated in FIGS. 2A-B. The plot in FIG.
2A is the initial data using a cell line control, and the plot in
FIG. 2B is an independent data set that uses a synthetic
standard.
[0063] When a cell line control is used, NSCLC and Normal Sample
results are significantly different from one another. Despite the
presence of some overlap, NSCLC samples consistently show ACTB to
FPR1 ratios and HNRNPA1 to FPR1 ratios that are significantly
greater than non-cancer samples when fit to a cell line
control.
[0064] When a synthetic RNA standard rather than a cell, line
control is used, similar results are obtained but the degree of
overlap is substantially decreased. This decreased overlap is due
to decreased variability in the standards resulting from reduced
numbers of serial dilutions (from 6 to 3). Each step of the serial
dilution may introduce error. In FIG. 2B, a simple line can be
drawn to separate all but one of the Normal synthetic standard
result ratios from all of the NSCLC results.
[0065] The results may also be interpreted as a single ratio
between a linear combination of the type #1 markers and a linear
combination of the type #2 markers. A decision rule may state that
any score above a given threshold indicates cancer, while a score
below the threshold indicates the lack of cancer. A synthetic
standard may be designed such that the coefficient on each marker
is 1, such that the score is calculated as:
Score=FRP1/(ACTB+HNRNPA1).
[0066] For example, transcript accumulation values for genes
selected from the lists above may be determined from a sample and
compared to levels determined from a set of synthetic standards
(i.e. in a serial dilution series) that span the range of values
that are typically obtained. For each gene, the transcript
accumulation level determined from a patient sample is compared to
the transcript accumulation level determined by performing a
regression analysis on a synthetic standard template to fit the
accumulation level values for each gene. The regression and fitted
values are obtained for each gene individually. Additional analysis
(i.e. calculating ratios) may be done once fitted values are
obtained.
[0067] These scores may be compared to threshold values, such, that
scores above a threshold are indicative of a heightened risk of
lung cancer as indicated by a patient sample.
[0068] It can be readily seen that, when this calculation is used
with a threshold of 1/2, it is the same as using the line drawn in
FIG. 2B. The correct concentrations for each standard, coefficients
and threshold may be determined by collecting data on a small set
of samples from both cancer and cancer-free patients, then using a
linear model to separate them. The linear model may be generated
via a statistical method such as logistic regression or support
vector machines with a linear kernel function, or the linear model
may be generated by inspection.
[0069] Exclusionary criteria may be implemented, such that any
sample that meets the exclusionary criteria has no result reported.
These exclusionary criteria may include other test preformed before
or after one of the described embodiments. The exclusionary
criteria may also be based on results of the test itself. For
example, in some embodiments very low quantities of the markers
indicate a degraded sample, and an unexpectedly large ratio between
two accumulation levels such as those of ACTB and HNRNPA1, for
example, may indicate that there is contamination. In some
embodiments a sample is excluded if the ratio of ACTB to HNRNPA1
differs by more than 10, 5, 4, 3, or 2-foled compared to the median
ratio of the accumulation levels of the genes. One example of a
plausible contamination source is that of lymphocytes in, the
plasma sample.
[0070] In some embodiments the method may involve a Statistical
Distance Determination. Because cfRNA from cancer cells may be
highly diluted, a method may be required to determine significant
changes in relative abundance. For this reason, in some
embodiments, the method determines the assay outcome (i.e.,
positive or negative result) based on statistical distances between
results as opposed to a fixed cutoff determined only through ROC
curves.
[0071] Based on the specificity, the results may be divided into
groups (high confidence, low confidence, etc.). This number may
also be transformed by some simple formula to create a numerical
score for confidence.
[0072] In some embodiments the method may involve Models and
Derivations for predicting the type of cancer present in a patient
based on results RNA expression in combination with demographic or
lifestyle attribute(s).
[0073] In some embodiments cfRNA levels may be assayed using
sequencing technology. Examples of sequencing technology include
but are not limited to one or more technologies such as
pyrosequencing, e.g., `the `454` method (Margulies et al., (2005)
Genome sequencing in microfabricated high-density picolitre
reactors. Nature 437:376-380; Ronaghi, et al. (1996) Real-time DNA
sequencing using detection of pyrophosphate release. Anal. Biochem.
242:84-89), `Solexa` or Illumina-type sequencing (Fedurco et al.,
(2006), BTA, a novel reagent for DNA attachment of glass and
efficient generation of solid-phase amplified DNA colonies. Nucleic
Acid Research 34, e22; Turcatti et al. (2008), A new class of
cleavable fluorescent nucleotides: synthesis and optimization as
reversible terminators for DNA sequencing by synthesis. Nucleic
Acid Research 36, e25), SOLiD sequencing technology (Shendure, J.
et al. (2005) Accurate multiplex polony sequencing of an evolved
bacterial genome. Science 309, 1728-1732; McKernan, K. et al,
(2006) Reagents, methods, and libraries for bead-based sequencing.
US patent application 20080003571), Heliscope Technology (Harris,
T. D. et al. (2008) Single-molecule DNA sequencing of a viral
genome. Science 320, 106-109), Ion Torrent Technology (Rothberg et
al., (2011) An integrated semiconductor device enabling non-optical
genome sequencing. Nature 475, 348-352), SMRT Sequencing Technology
(Pacific Biosciences), or GridION nanopore-based sequencing (Oxford
Nanopore Technologies;
http://www.nanoporetech.com/technology/the-gridion-system/the-gridion-sys-
tem). In some embodiments any number of so-called `next generation`
DNA sequencing methods may be used, as described in Shendure and
Ji, "Next-generation DNA sequencing", Nature Biotechnology
26(10):1135-1145 (2008) or in other art available to one of skill
in the art. Other methods for the determination of DNA sequence are
also known in the art, and embodiments disclosed herein are not
limited to any particular method of determining base identity at a
particular locus to the exclusion of any other method.
[0074] In some embodiments, the cfRNA levels may be assayed via
hybridization to a microarray, nCounter or similar. For example,
one class of arrays commonly used in differential expression
studies includes microarrays or oligonucleotide arrays. These
arrays utilize a large number of probes that are synthesized
directly on a substrate and are used to interrogate complex RNA or
message populations based on the principle of complementary
hybridization. Typically, these microarrays provide sets of 16 to
20 oligonucleotide probe pairs of relatively small length
(20mers-25mers) that span a selected region of a gene or nucleotide
sequence of interest. The probe pairs used in the oligonucleotide
array may also include perfect match and mismatch probes that are
designed to hybridize to the same RNA or message strand. The
perfect match probe contains a known sequence that is fully
complementary to the message of interest while the mismatch probe
is similar to the perfect match probe with respect to its sequence
except that it contains at least one mismatch nucleotide which
differs from the perfect match probe. During expression analysis,
the hybridization efficiency of messages from a sample nucleotide
population are assessed with respect to the perfect match and
mismatch probes in order to validate and quantitate the levels of
expression for many messages simultaneously. In some embodiments an
entire gene array is printed to a microarray. In some embodiments a
subset of genes comprising FPR1 and at least one of ACTB and
HNRNPA1 is included on a microarray. In some embodiments a
microarray comprises at least FPR1, ACTB and HNRNPA1.
[0075] A device such as an nCounter, offered by Nanostring
technologies, for example, may be used to facilitate analysis. An
nCounter Analysis System is an integrated system comprising a fully
automated prep station, a digital analyzer, the CodeSet (molecular
barcodes) and all of the reagents and consumables needed to perform
the analysis. Analysis on the nCounter system consists of
in-solution hybridization, post-hybridization processing, digital
data acquisition, and normalization in one simple workflow. In some
embodiments the process is automated. In some embodiments custom or
pre-designed sets of barcoded probes may be pre-mixed with a
comprehensive set of system controls as part of said analysis.
[0076] A number of methods of and devices for obtaining the cfRNA
transcript accumulation level, data necessary to perform the
methods and for use with the compositions and kits disclosed
herein, and no single data accumulation method or device should be
seen as limiting.
EXAMPLES
Example 1
[0077] Plasma was collected from, patients known to have non-small
cell lung cancer ("NSCLC") and patients without any known lung
cancer (putatively "cancer free" individuals). There is some
possibility that patients without any known lung cancer may in fact
have an otherwise undetected cancer. The presence of these patients
will lead to an over-estimation of the false positive rate for this
test (because "false positives" from "healthy individuals" may in
fact represent the presence of cancer in these individuals). After
removing plasma that had obvious issues, such as orange color or
turbidity, 25 cancer-free and 26 NSCLC plasma samples remained. The
following stages of cancer were present from the 26 NSCLC patients:
8 stage I, 6 stage II, 5 stage III, and 7 stage unknown. The plasma
was extracted with the QIAamp circulating nucleic acid kit (Qiagen
#55114). The Quantifast Probe RT-PCR Plus Kit (Qiagen #204484) was
used along with the previously described primers and probes for
FPR1, ACTB, and HNRNPA1 to conduct quantitative Taqman PCR.
Universal Human Reference RNA (Agilent #740000) was used as a
standard, and regression was used to estimate quantities of each
gene in the sample (FIG. 2A). Samples with a ratio of ACTB/HNRNPA1
above 75 were eliminated from the final results, and given a result
of "no result", The following results were obtained. Note that the
x-axis is the ratio of FPR1/ACTB and the Y-axis is the ratio of
FPR1/HNRNPA1.
[0078] As shown in FIG. 1A, FPR1 median accumulation levels
normalized against accumulation levels measured in Universal Human
Reference RNA differed by about 100-fold between Normal and Tumor
patient populations. Similarly, the upper limit of the 25% quartile
of the Normal patent population was measurably below the lower
limit 75% quartile of the measurements for the Tumor patient
population. The highest, rarest extreme Normal measurements
occasionally were as high as, but no higher than, the Tumor patient
median values.
[0079] This result shows that measuring the circulating free RNA
levels of FPR1 allows one to predict the presence of cancer in a
patient.
[0080] As shown in FIG. 1B, ACTB median accumulation levels did not
differ significantly between Normal and Tumor patient populations.
There was also substantial overlap at the 25%-75% quartile range
and among the extreme outliers in accumulation levels between
Normal, and Tumor patient populations.
[0081] Similarly, yin FIG. 1C, HNRNPA1 median accumulation levels
did not differ significantly between Normal and Tumor patient
populations. There was also substantial overlap at the 25%-75%
quartile range and among the extreme outliers in accumulation
levels between Normal and Tumor patient populations.
[0082] This result, demonstrates the suitability of ACTB and
HNRNPA1 as subset #2 constituents or as reference standards.
[0083] As shown in FIG. 2A, when a cell line control was used,
Tumor and Normal Sample results were significantly different from
one another. Despite the presence of some overlap, NSCLC samples
consistently showed FPR1 to ACTB ratios and FPR1 to HNRNPA1 ratios
that were in general significantly greater than the corresponding
values obtained for a cell line control.
[0084] This experiment shows that comparing the circulating free
RNA levels of FPR1 to the circulating free RNA levels of HNRNPA1 or
ACTB allows one to predict the presence of cancer in a patient.
Example 2
[0085] A second set of 6 NSCLC and 7 Normal patient samples were
assayed using a protocol similar to that discussed in Example 1.
Two technical modifications were made: plasma was extracted via the
QIAsymphony DSP Virus/Pathogen Midi Kit (Qiagen 937055) and a
synthetic standard as described above was used. Additional samples
were excluded after a screen for obvious issues such as cloudiness
or discoloration. The ratios of the fitted values for each gene
were determined, and are shown in FIG. 2B. The X and Y axis are the
same ratios as previously described.
[0086] As shown in FIG. 2B, when a synthetic standard was used,
NSCLC and Normal Sample results were significantly different from
one another. NSCLC samples consistently showed ACTB to FPR1 ratios
and HNRNPA1 to FPR1 ratios that were significantly greater than the
corresponding values obtained, for plasma obtained from cancer-free
individuals. A simple line can be drawn to separate all but one of
the Normal synthetic standard result ratios from all of the NSCLC
results.
[0087] While it may initially appear that there is a wider gap
between the NSCLC and cancer-free group, this may be due to the
smaller number of points. As fewer points are present, extreme
values are less probable. Some reduction in, variability may also
be due to the smaller number of dilutions used creation of the
synthetic standard. This is enabled by the ability to fine tune the
concentration of each gene individually in the standard.
Example 3
[0088] Plasma was collected from a patient suspected of having lung
cancer. FPR1, ACTB and HNRNPA1 transcript accumulation levels in
the cfRNA population of said patient were determined from the
patient's plasma as described above. Patient FPR1 accumulation
levels were compared to levels known to correspond with healthy
individuals and with individuals that have cancer, for example, the
accumulation levels indicated in FIG. 1A. Other reference measures
of FPR1 accumulation in cfRNA of healthy and cancer-positive
individuals could also have been used. An arbitrary value of 100
was assigned to the full concentration of the standard for each
gene, with the two additional dilutions used to create the standard
curve being 10 and 1 arbitrary units. Sample 171 was collected from
a patient known to have NSCLC. The values of 18.14, 26.64 and 5.38
were obtained for FPR1, ACTB and HNRNPA1 respectively. The score
was calculated as 18.14/(26.64+5.38)=0.567 was obtained. Because
this value is greater than 0.5, a positive result for NSCLC was
obtained.
[0089] Plasma was collected from patient 164 with no evidence of
cancer. Transcript accumulation levels of FPR1, ACTB and HNRNPA1
were obtained as above. The values of 3.53, 12.2 and 2.87 were
obtained for FPR1, ACTB and HNRNPA1 respectively. The score is
calculated as 3.53/(12.2+2.87)=0.234 was obtained. Because this
value is less than 0.5, a negative result for NSCLC was
obtained.
[0090] This experiment shows that determining the circulating free
RNA levels of FPR1, ACTB and HNRNPA1 allows one to predict the
presence of cancer, and particularly lung cancer, in a patient.
Example 4
[0091] Plasma is collected from a patient suspected of having lung
cancer. FPR1 transcript accumulation levels in the cfRNA population
of said patient are determined from the patient's plasma as
described above. Patient FPR1 accumulation levels are compared to
levels known to correspond with healthy individuals and with
individuals that have cancer, for example, the accumulation levels
indicated in FIG. 1A. Other reference measures of FPR1 accumulation
in cfRNA of healthy and cancer-positive individuals may be used.
Levels are found to be 60,000 FPR1 molecules per mL, which
corresponds to the median accumulation level observed for Tumor
patients but is at or above the extreme highest measured value for
Normal patients.
[0092] This FPR1 accumulation level indicates with a high degree of
confidence that a NSCLC cell population is present in the
patient.
[0093] Plasma is collected from a second patient suspected of
having lung cancer. FPR1 transcript accumulation levels in the
cfRNA population of said patient are determined and compared as
above. Levels are found to be 600 FPR1 molecules per mL, which
corresponds to the median accumulation level observed for Normal
patients but is at or below the extreme lowest measured value for
Tumor patients. This FPR1 accumulation level indicates with a high
degree of confidence that the patient is free of a cancerous or
precancerous cell population detectable through this method.
[0094] This experiment shows that determining the circulating free
RNA levels of FPR1 allows one to predict the presence of cancer in
a patient.
Sequence CWU 1
1
12118DNAHomosapiens 1tgacggtgag aggcatca 18223DNAHomosapiens
2cggttcatca ttggcttcag cgc 23322DNAHomosapiens 3ggtggcaata
agcccataac tg 22418DNAHomosapiens 4aggccaaccg cgagaaga
18527DNAHomosapiens 5tgacccagat catgtttgag accttca
27622DNAHomosapiens 6tgccatccta aaagccaccc ca 22721DNAHomosapiens
7gggctttgcc tttgtaacct t 21823DNAHomosapiens 8tgacgaccat gactccgtgg
ata 23922DNAHomosapiens 9tgtggccatt cacagtatgg ta
221096RNAHomosapiens 10cauguugacg gugagaggca ucauccgguu caucauuggc
uucagcgcac ccauguccau 60cguugcuguc aguuaugggc uuauugccac caagau
961192RNAHomosapiens 11ccccaaggcc aaccgcgaga agaugaccca gaucauguuu
gagaccuuca acaccccagc 60cauguacguu gcuauccagg cugugcuauc cc
921293RNAHomosapiens 12aaaaggggcu uugccuuugu aaccuuugac gaccaugacu
ccguggauaa gauugucauu 60cagaaauacc auacugugaa uggccacaac ugu 93
* * * * *
References