U.S. patent application number 17/096817 was filed with the patent office on 2022-03-24 for non-invasive blood based monitoring of genomic alterations in cancer.
This patent application is currently assigned to Dana-Farber Cancer Institute, Inc.. The applicant listed for this patent is Dana-Farber Cancer Institute, Inc.. Invention is credited to Pasi A. Janne, Yanan Kuang, Geoffrey Oxnard, Cloud P. Paweletz.
Application Number | 20220090203 17/096817 |
Document ID | / |
Family ID | |
Filed Date | 2022-03-24 |
United States Patent
Application |
20220090203 |
Kind Code |
A9 |
Janne; Pasi A. ; et
al. |
March 24, 2022 |
NON-INVASIVE BLOOD BASED MONITORING OF GENOMIC ALTERATIONS IN
CANCER
Abstract
The invention provides methods to monitor cell free nucleic
acids. The method comprises obtaining a plasma sample from a
subject known to have a cancer characterized by a pair of mutually
exclusive mutations specific to the cancer; isolating cell free
nucleic acids from the plasma sample obtained from the subject;
measuring the amount a housekeeping gene and/or total DNA in the
cell free nucleic acids isolated from the plasma sample to confirm
that the amount of housekeeping gene and/or total DNA in the sample
is within a selected range; measuring the amount of a first of the
pair of mutually exclusive mutations specific to the cancer in the
cell free nucleic acids isolated from the plasma sample; and
indicating in a report that the subject has the first mutation when
(a) the amount of the housekeeping gene and/or total DNA in the
cell free nucleic acids isolated from the plasma sample is within
the selected range and (b) the amount of the first mutation is
increased as compared to a control amount, wherein the control
amount is determined by measuring the apparent amount of the first
mutation in control cell free nucleic acids isolated from plasma
samples obtained from control subjects known to have the second of
the pair of mutually exclusive mutations specific to the cancer
using measuring conditions substantially the same as those used to
measure the amount of the first mutation in the cell free nucleic
acids isolated from the plasma sample from the subject.
Inventors: |
Janne; Pasi A.; (Needham,
MA) ; Paweletz; Cloud P.; (Boston, MA) ;
Oxnard; Geoffrey; (Arlington, MA) ; Kuang; Yanan;
(Belmont, MA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Dana-Farber Cancer Institute, Inc. |
Boston |
MA |
US |
|
|
Assignee: |
Dana-Farber Cancer Institute,
Inc.
Boston
MA
|
Prior
Publication: |
|
Document Identifier |
Publication Date |
|
US 20210207223 A1 |
July 8, 2021 |
|
|
Appl. No.: |
17/096817 |
Filed: |
November 12, 2020 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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14897269 |
Dec 10, 2015 |
10865451 |
|
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PCT/US2014/041871 |
Jun 11, 2014 |
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17096817 |
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61889148 |
Oct 10, 2013 |
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61833556 |
Jun 11, 2013 |
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International
Class: |
C12Q 1/6886 20060101
C12Q001/6886; C12Q 1/6827 20060101 C12Q001/6827; A61K 31/437
20060101 A61K031/437; A61K 31/517 20060101 A61K031/517 |
Goverment Interests
FEDERALLY SPONSORED RESEARCH
[0002] This invention was made with government support under grant
numbers P50 CA090578 and R01 CA135257 awarded by the National
Cancer Institute and the National Institutes of Health. The
government has certain rights in the invention.
Claims
1. A method to monitor cell free DNA comprising: (i) obtaining a
body fluid sample from a subject known to have a cancer
characterized by a pair of mutually exclusive mutations specific to
the cancer; (ii) isolating cell free nucleic acids from the body
fluid sample obtained from the subject; (iii) measuring the amount
of a housekeeping gene and/or total DNA in the cell free nucleic
acids isolated from the body fluid sample, wherein the amount of a
housekeeping gene and/or total DNA in the sample correlates to the
amount of Line 1 and the amount of Line 1 is within a range of
3,000-700,000 pg/.mu.L; (iv) measuring the amount of a first of the
pair of mutually exclusive mutations specific to the cancer in the
cell free nucleic acids isolated from the body fluid sample; and
(v) indicating in a report that the subject has the first of the
pair of mutually exclusive mutations when (a) the amount of the
housekeeping gene and/or total DNA in the cell free nucleic acids
isolated from the body fluid sample correlates to the amount of
Line 1 and the amount of Line 1 is within the range of
3,000-700,000 pg/.mu.L and (b) the amount of the first of the pair
of mutually exclusive mutations is increased as compared to a
control amount, wherein the control amount is the apparent amount
of the first of the pair of mutually exclusive mutations in control
cell free nucleic acids isolated from body fluid samples obtained
from control subjects known to have the second of the pair of
mutually exclusive mutations specific to the cancer using measuring
conditions substantially the same as those used to measure the
amount of the first of the pair of mutually exclusive mutations in
the cell free nucleic acids isolated from the body fluid sample
from the subject.
2. The method of claim 1, wherein the measuring of: the first of
the pair of mutually exclusive mutations specific to the cancer in
the cell free nucleic acids isolated from the body fluid sample
obtained from the subject is performed by quantitative PCR,
microarrays, Next-generation sequencing, chemiluminescence methods,
fluorescent methods, digital detection, or mass spectrometry
(MALDI-TOF).
3. The method of claim 1, wherein the cancer is lung cancer or
colon cancer.
4. The method of claim 3, wherein the pair of mutually exclusive
mutations is an epidermal growth factor receptor (EGFR) mutation
and a Rat sarcoma (RAS) mutation, wherein the pair of mutually
exclusive mutations comprises an epidermal growth factor receptor
(EGFR) mutation and a v-Ki-ras2 Kirsten rat sarcoma viral oncogene
homolog (KRAS) mutation, or wherein the pair of mutually exclusive
mutations comprises a v-raf murine sarcoma viral oncogene homolog
B1 (BRAF) mutation and a Rat sarcoma (RAS) mutation.
5. (canceled)
6. The method of claim 4, wherein the EGFR mutation is selected
from the group consisting of: L858R, T790M, L861Q, G719S, del 19
and exon 20 insertions.
7. The method of claim 4, wherein the KRAS mutation is G12C.
8-14. (canceled)
15. The method of claim 1, wherein the amount of the first of the
pair of mutually exclusive mutations specific to the cancer is
measured by digital droplet PCR.
16. (canceled)
17. The method of claim 1, wherein the amount of the first of the
pair of mutually exclusive mutations is measured before and after
administration of an anti-cancer therapy to the subject.
18. The method of claim 1, wherein step (i)-step (iv) are repeated
so as to monitor the subject's amount of the first of the pair of
mutually exclusive mutations over time.
19. The method of claim 18, wherein a decrease in amount of the
first of the pair of mutually exclusive mutations indicates that
the cancer is stabilizing or decreasing, or wherein an increase in
amount of the first of the pair of mutually exclusive mutations
indicates that the cancer is increasing.
20-22. (canceled)
23. A method to monitor cell free DNA comprising: (i) obtaining a
body fluid sample from a subject known to have a cancer
characterized by a pair of mutually exclusive mutations specific to
the cancer; (ii) isolating cell free nucleic acids from the body
fluid sample obtained from the subject; (iii) measuring the amount
of a housekeeping gene and/or total DNA in the cell free nucleic
acids isolated from the body fluid sample, wherein the amount of
the housekeeping gene and/or total DNA in the sample correlates to
the amount of Line 1 and the amount of Line 1 is within a range of
3,000-700,000 pg/.mu.L; (iv) measuring the amount of a first of the
pair of mutually exclusive mutations specific to the cancer in the
cell free nucleic acids isolated from the body fluid sample; and
(v) measuring the apparent amount of the first of the pair of
mutually exclusive mutations in control cell free nucleic acids
isolated from body fluid samples obtained from control subjects
known to have the second of the pair of mutually exclusive
mutations specific to the cancer using measuring conditions
substantially the same as those used to measure the amount of the
first of the pair of mutually exclusive mutations in the cell free
nucleic acids isolated from the body fluid sample from the subject;
and (vi) indicating in a report that the subject has the first of
the pair of mutually exclusive mutations when (a) the amount of the
housekeeping gene and/or total DNA in the cell free nucleic acids
isolated from the body fluid sample correlates to the amount of
Line 1 and the amount of Line 1 is within the range of
3,000-700,000 pg/.mu.L and (b) the amount of the first of the pair
of mutually exclusive mutations is increased as compared to the
apparent amount of the first of the pair of mutually exclusive
mutations in control cell free nucleic acids isolated from body
fluid samples obtained from control subjects.
24. (canceled)
25. A method to treat cancer comprising: (i) obtaining a body fluid
sample from a subject known to have a cancer characterized by a
pair of mutually exclusive mutations specific to the cancer; (ii)
isolating cell free nucleic acids from the body fluid sample
obtained from the subject; (iii) measuring the amount of a
housekeeping gene and/or total DNA in the cell free nucleic acids
isolated from the body fluid sample, wherein the amount of
housekeeping gene and/or total DNA in the sample correlates to the
amount of Line 1 and the amount of Line 1 is within a range of
3,000-700,000 pg/.mu.L; (iv) measuring the amount of a first of the
pair of mutually exclusive mutations specific to the cancer in the
cell free nucleic acids isolated from the body fluid sample; (v)
measuring the apparent amount of the first of the mutually
exclusive mutations in control cell free nucleic acids isolated
from body fluid samples obtained from control subjects known to
have the second of the pair of mutually exclusive mutations
specific to the cancer using measuring conditions substantially the
same as those used to measure the amount of the first of the
mutually exclusive mutations in the cell free nucleic acids
isolated from the body fluid sample from the subject; and (vi)
treating the subject with an anti-cancer therapy when (a) the
amount of the housekeeping gene and/or total DNA in the cell free
nucleic acids isolated from the body fluid sample is within the
range of 3,000-700,000 pg/.mu.L and (b) the amount of the first of
the pair of mutually exclusive mutations is increased as compared
to the apparent amount of the first of the pair of mutually
exclusive mutations in control cell free nucleic acids isolated
from body fluid samples obtained from control subjects.
26-41. (canceled)
42. The method of claim 25, wherein step (i)-step (iv) are repeated
so as to monitor the subject's amount of the first of the pair of
mutually exclusive mutations over time.
43. The method of claim 42, wherein administration of the
anti-cancer therapy is maintained when the amount of the mutation
decreases over time, or wherein the anti-cancer therapy is
administered at a higher dosage or is changed when the amount of
the mutation increases over time.
44-46. (canceled)
47. A method comprising: (i) administering an anti-cancer therapy
to a subject known to have a cancer characterized by a pair of
mutually exclusive mutations specific to the cancer; (ii) obtaining
a body fluid sample from the subject; (iii) isolating cell free
nucleic acids from the body fluid sample obtained from the subject;
(iv) measuring the amount of a housekeeping gene and/or total DNA
in the cell free nucleic acids isolated from the body fluid sample
to confirm that the amount of the housekeeping gene and/or total
DNA in the sample correlates to the amount of Line and the amount
of Line 1 is within a range of 3,000-700,000 pg/.mu.L; (v)
measuring the amount of a first of the pair of mutually exclusive
mutations specific to the cancer in the cell free nucleic acids
isolated from the body fluid sample; and (vi) measuring the
apparent amount of the first of the pair of mutually exclusive
mutations in control cell free nucleic acids isolated from body
fluid samples obtained from control subjects known to have the
second of the pair of mutually exclusive mutations specific to the
cancer using measuring conditions substantially the same as those
used to measure the amount of the first of the pair of mutually
exclusive mutations in the cell free nucleic acids isolated from
the body fluid sample from the subject.
48-68. (canceled)
69. The method of claim 15, wherein the amount of the first of the
pair of mutually exclusive mutations specific to the cancer is
determined by: preparing at least 2 serial dilutions of the cell
free nucleic acids isolated from the body fluid sample; measuring
the amount of the first of the pair of mutually exclusive mutations
in the at least 2 serial dilutions using digital droplet PCR; and
evaluating linearity of the measured dilutions to confirm accuracy
of the method.
70. The method of claim 18, wherein an increase in amount of the
mutation indicates that the cancer is increasing.
71. The method of claim 18, wherein the subject's amount of the
first of the pair of mutually exclusive mutations is measured: (a)
in a first sample obtained from the subject before the subject
received an anti-cancer therapy; and (b) in a second sample
obtained from the subject after the subject received an anti-cancer
therapy.
72. The method of claim 23, wherein the measuring of: (a) the first
of the pair of mutually exclusive mutations specific to the cancer
in the cell free nucleic acids isolated from the body fluid sample
obtained from the subject and (b) the apparent amount of the first
of the pair of mutually exclusive mutations in cell free nucleic
acids isolated from control body fluid samples obtained from
control subjects known to have the second of the pair of mutually
exclusive mutations specific to the cancer is performed by
microarrays, Next-generation sequencing, chemiluminescence methods,
fluorescent methods, digital detection, or mass spectrometry
(MALDI-TOF).
73. A method to monitor cell free DNA comprising: (i) obtaining a
body fluid sample from a subject known to have a cancer
characterized by a pair of mutually exclusive mutations specific to
the cancer; (ii) isolating cell free nucleic acids from the body
fluid sample obtained from the subject; (iii) measuring the amount
of a housekeeping gene and/or total DNA in the cell free nucleic
acids isolated from the body fluid sample, wherein the amount of a
housekeeping gene and/or total DNA in the sample correlates to the
amount of Line and the amount of Line 1 is within a range of
3,000-700,000 pg/.mu.L of body fluid; (iv) measuring the amount of
the first of the pair of mutually exclusive mutations specific to
the cancer in the cell free nucleic acids isolated from the body
fluid sample; and (v) measuring the apparent amounts of the first
of the pair of mutually exclusive mutations in control cell free
nucleic acids isolated from body fluid samples obtained from a
plurality of control subjects known to have the second of the pair
of mutually exclusive mutations specific to the cancer using
measuring conditions substantially the same as those used to
measure the amount of the first of the pair of mutually exclusive
mutations in the cell free nucleic acids isolated from the body
fluid sample from the subject; and (vi) indicating a result in a
report wherein: the amount measured in (iv) is greater than the
highest amount measured in (v) and indicating in a report that the
body fluid sample from the subject has the first of the pair of
mutually exclusive mutations; or the amount measured in (iv) is
less than the highest amount measured in (v) and indicating a
report that the body fluid sample from the subject does not have
the first of the pair of mutually exclusive mutations.
74. The method of claim 73, wherein the measuring of: the first of
the pair of mutually exclusive mutations specific to the cancer in
the cell free nucleic acids isolated from the body fluid sample
obtained from the subject is performed by quantitative PCR,
microarrays, Next-generation sequencing, chemiluminescence methods,
fluorescent methods, digital detection, or mass spectrometry
(MALDI-TOF).
75. The method of claim 73, wherein the cancer is lung cancer or
colon cancer.
76. The method of claim 73, wherein the pair of mutually exclusive
mutations is an epidermal growth factor receptor (EGFR) mutation
and a Rat sarcoma (RAS) mutation, or wherein the pair of mutually
exclusive mutations comprises an epidermal growth factor receptor
(EGFR) mutation and a v-Ki-ras2 Kirsten rat sarcoma viral oncogene
homolog (KRAS) mutation, or wherein the pair of mutually exclusive
mutations comprises a v-raf murine sarcoma viral oncogene homolog
B1 (BRAF) mutation and a Rat sarcoma (RAS) mutation.
77. The method of claim 76, wherein the EGFR mutation is selected
from the group consisting of: L858R, T790M, L861Q, G719S, del 19
and exon 20 insertions.
78. The method of claim 73, wherein the amount of the first of the
pair of mutually exclusive mutations is measured before and after
administration of an anti-cancer therapy to the subject.
79. The method of claim 73, wherein step (i)-step (iv) are repeated
so as to monitor the subject's amount of the first of the pair of
mutually exclusive mutations over time.
Description
RELATED APPLICATIONS
[0001] This application is a continuation of and claims priority
under 35 U.S.C. .sctn. 120 to U.S. patent application U.S. Ser. No.
14/897,269, filed Dec. 10, 2015, which is a national stage filing
under 35 U.S.C. .sctn. 371 of International PCT Application,
PCT/US2014/041871, filed Jun. 11, 2014, and entitled "NON-INVASIVE
BLOOD BASED MONITORING OF GENOMIC ALTERATIONS IN CANCER," which
claims priority under 35 U.S.C. .sctn. 119(e) to U.S. Provisional
Patent Applications 61/889,148, filed on Oct. 10, 2013 and
61/833,556, filed on Jun. 11, 2013, each of which are incorporated
herein in their entirety.
FIELD OF THE INVENTION
[0003] The present invention relates in general to cancer. More
specifically, the invention relates to methods monitoring cell free
DNA for performing disease monitoring and pharmacodynamic
assessment of drug efficacy.
BACKGROUND OF THE INVENTION
[0004] Cancer remains a major health concern. Despite increased
understanding of many aspects of cancer, the methods available for
its treatment continue to have limited success. A major limitation
in current cancer therapy is a lack of understanding of the
molecular changes in cancers in response to therapies. This is
particularly exemplified for cancers such as epidermal growth
factor receptor (EGFR) mutant lung cancer or BRAF mutant melanoma,
where despite initial dramatic clinical efficacy of erlotinib or
vermurafenib, drug resistance to these targeted therapies
ultimately develops in all patients. An understanding of when and
how this occurs may help guide subsequent therapeutic choices.
[0005] The challenges of genotype-directed cancer care are mostly
driven by the inability to get repeat biopsies from the same
patients. Thus, performing genotyping of tumors using body fluids,
such as blood is desirable. However, blood has very low
concentrations of the DNA fragments of interest (that is derived
from the tumor), requiring high sensitivity assays. These assays
have a number of limitations including low specificity, i.e., false
positives. Another challenge with high sensitivity assays is
identifying a "gold standard" wild-type population given that
conventional tumor genotyping does have a chance of being falsely
negative. Accordingly, there is a need in the art for
high-sensitivity, high-specificity assays for the detection of
molecular indicia of cancer.
SUMMARY OF THE INVENTION
[0006] The invention, relates in some aspects to the finding that
cell free nucleic acids into body fluids by tumor cells have
diagnostic and prognostic utility. The inventors of the present
invention have generated a control platform that allows an accurate
determination of whether a person carries the mutation of interest,
or whether the result obtained is an artifact of the measuring
assay. This platform is based on two concepts: (i) a quality
control step and (ii) a `gold standard` control population.
According to one aspect of the invention, a method to monitor cell
free DNA is provided. The method comprises obtaining a plasma
sample from a subject known to have a cancer characterized by a
pair of mutually exclusive mutations specific to the cancer;
isolating cell free nucleic acids from the plasma sample obtained
from the subject; measuring the amount a housekeeping gene and/or
total DNA in the cell free nucleic acids isolated from the plasma
sample to confirm that the amount of housekeeping gene and/or total
DNA in the sample is within a selected range; measuring the amount
of a first of the pair of mutually exclusive mutations specific to
the cancer in the cell free nucleic acids isolated from the plasma
sample; and indicating in a report that the subject has the first
mutation when (a) the amount of the housekeeping gene and/or total
DNA in the cell free nucleic acids isolated from the plasma sample
is within the selected range and (b) the amount of the first
mutation is increased as compared to a control amount, wherein the
control amount is determined by measuring the apparent amount of
the first mutation in control cell free nucleic acids isolated from
plasma samples obtained from control subjects known to have the
second of the pair of mutually exclusive mutations specific to the
cancer using measuring conditions substantially the same as those
used to measure the amount of the first mutation in the cell free
nucleic acids isolated from the plasma sample from the subject.
[0007] According to some aspects of the invention, a method to
monitor cell free DNA is provided. The method comprises obtaining a
plasma sample from a subject known to have a cancer characterized
by a pair of mutually exclusive mutations specific to the cancer;
isolating cell free nucleic acids from the plasma sample obtained
from the subject; measuring the amount a housekeeping gene and/or
total DNA in the cell free nucleic acids isolated from the plasma
sample to confirm that the amount of housekeeping gene and/or total
DNA in the sample is within a selected range; measuring the amount
of a first of the pair of mutually exclusive mutations specific to
the cancer in the cell free nucleic acids isolated from the plasma
sample; and measuring the apparent amount of the first mutation in
control cell free nucleic acids isolated from plasma samples
obtained from control subjects known to have the second of the pair
of mutually exclusive mutations specific to the cancer using
measuring conditions substantially the same as those used to
measure the amount of the first mutation in the cell free nucleic
acids isolated from the plasma sample from the subject. In some
embodiments, the method further comprises indicating in a report
that the subject has the first mutation when (a) the amount of the
housekeeping gene and/or total DNA in the cell free nucleic acids
isolated from the plasma sample is within the selected range and
(b) the amount of the first mutation is increased as compared to a
control amount.
[0008] In some embodiments, the amount of the first mutation is
measured before and after administration of an anti-cancer therapy
to the subject. In some embodiments, the sample collection,
isolation and measuring steps are repeated so as to monitor the
subject's amount of the first mutation over time. In some
embodiments, a decrease in amount of the mutation indicates that
the cancer is stabilizing or decreasing. In some embodiments, an
increase in amount of the mutation indicates that the cancer is
increasing. In some embodiments, the subject's amount of the first
mutation is measured: (a) in a first sample obtained from the
subject before the subject received an anti-cancer therapy; and (b)
in a second sample obtained from the subject after the subject
received an anti-cancer therapy.
[0009] According to some aspects of the invention, a method to
treat cancer is provided. The method comprises obtaining a plasma
sample from a subject known to have a cancer characterized by a
pair of mutually exclusive mutations specific to the cancer;
isolating cell free nucleic acids from the plasma sample obtained
from the subject; measuring the amount a housekeeping gene and/or
total DNA in the cell free nucleic acids isolated from the plasma
sample to confirm that the amount of housekeeping gene and/or total
DNA in the sample is within a selected range; measuring the amount
of a first of the pair of mutually exclusive mutations specific to
the cancer in the cell free nucleic acids isolated from the plasma
sample; measuring the apparent amount of the first mutation in
control cell free nucleic acids isolated from plasma samples
obtained from control subjects known to have the second of the pair
of mutually exclusive mutations specific to the cancer using
measuring conditions substantially the same as those used to
measure the amount of the first mutation in the cell free nucleic
acids isolated from the plasma sample from the subject; and
treating the subject with an anti-cancer therapy when (a) the
amount of the housekeeping gene and/or total DNA in the cell free
nucleic acids isolated from the plasma sample is within the
selected range and (b) the amount of the first mutation is
increased as compared to a control amount.
[0010] In some embodiments, the amount of the first mutation is
measured before and after administration of the anti-cancer therapy
to the subject. In some embodiments, the sample collection,
isolation and measuring steps are repeated so as to monitor the
subject's amount of the first mutation over time. In some
embodiments, administration of the anti-cancer therapy is
maintained when the amount of the mutation decreases over time. In
some embodiments, the anti-cancer therapy is administered at a
higher dosage or is changed when the amount of the mutation
increases over time. In some embodiments, the subject's amount of
the first mutation is measured: (a) in a first sample obtained from
the subject before the subject received the anti-cancer therapy;
and (b) in a second sample obtained from the subject after the
subject received the anti-cancer therapy.
[0011] According to some aspects of the invention, a method to
monitor efficacy of an anti-cancer therapy is provided. The method
comprises administering an anti-cancer therapy to a subject known
to have a cancer characterized by a pair of mutually exclusive
mutations specific to the cancer; obtaining a plasma sample from
the subject; isolating cell free nucleic acids from the plasma
sample obtained from the subject; measuring the amount a
housekeeping gene and/or total DNA in the cell free nucleic acids
isolated from the plasma sample to confirm that the amount of
housekeeping gene and/or total DNA in the sample is within a
selected range; measuring the amount of a first of the pair of
mutually exclusive mutations specific to the cancer in the cell
free nucleic acids isolated from the plasma sample; and measuring
the apparent amount of the first mutation in control cell free
nucleic acids isolated from plasma samples obtained from control
subjects known to have the second of the pair of mutually exclusive
mutations specific to the cancer using measuring conditions
substantially the same as those used to measure the amount of the
first mutation in the cell free nucleic acids isolated from the
plasma sample from the subject.
[0012] In some embodiments, the amount of the first mutation is
measured before and after administration of the anti-cancer therapy
to the subject. In some embodiments, the sample collection,
isolation and measuring steps are repeated so as to monitor the
subject's amount of the first mutation over time. In some
embodiments, the anti-cancer therapy is efficacious when the amount
of the mutation decreases over time. In some embodiments, the
anti-cancer therapy is not efficacious when the amount of the
mutation increases over time. In some embodiments, the subject's
amount of the first mutation is measured: (a) in a first sample
obtained from the subject before the subject received the
anti-cancer therapy; and (b) in a second sample obtained from the
subject after the subject received the anti-cancer therapy.
[0013] The following embodiments apply equally to the various
aspects of the invention set forth herein unless indicated
otherwise.
[0014] In some embodiments, the measuring of: (a) the first of the
pair of mutually exclusive mutations specific to the cancer in the
cell free nucleic acids isolated from the plasma sample obtained
from the subject and (b) the apparent amount of the first mutation
in cell free nucleic acids isolated from control plasma samples
obtained from control subjects known to have the second of the pair
of mutually exclusive mutations specific to the cancer is performed
by quantitative PCR.
[0015] In some embodiments, the cancer is lung cancer. In some
embodiments, the pair of mutually exclusive mutations comprises an
epidermal growth factor receptor (EGFR) mutation and a Rat sarcoma
(RAS) mutation. In some embodiments, the pair of mutually exclusive
mutations comprises an epidermal growth factor receptor (EGFR)
mutation and a v-Ki-ras2 Kirsten rat sarcoma viral oncogene homolog
(KRAS) mutation. In some embodiments, the EGFR mutation is selected
from the group consisting of: L858R, T790M, L861Q, G719S, del 19
and exon 20 insertions. In some embodiments, the KRAS mutation is
G12C.
[0016] In some embodiments, the cancer is colon cancer. In some
embodiments, the pair of mutually exclusive mutations comprises a
v-raf murine sarcoma viral oncogene homolog B1 (BRAF) mutation and
a Rat sarcoma (RAS) mutation. In some embodiments, the pair of
mutually exclusive mutations comprises a v-raf murine sarcoma viral
oncogene homolog B1 (BRAF) mutation and a v-Ki-ras2 Kirsten rat
sarcoma viral oncogene homolog (KRAS) mutation. In some
embodiments, the BRAF mutation is V600E.
[0017] In some embodiments, the cancer is a melanoma. In some
embodiments, the pair of mutually exclusive mutations comprises a
v-raf murine sarcoma viral oncogene homolog B1 (BRAF) mutation and
a Rat sarcoma (RAS) mutation. In some embodiments, the pair of
mutually exclusive mutations comprises a v-raf murine sarcoma viral
oncogene homolog B1 (BRAF) mutation and a neuroblastoma RAS viral
(v-ras) oncogene homolog (NRAS) mutation.
[0018] In some embodiments, the amount of the first of the pair of
mutually exclusive mutations specific to the cancer is measured by
digital droplet PCR. In some embodiments, the amount of the first
of the pair of mutually exclusive mutations specific to the cancer
is determined by: preparing at least 2 serial dilutions of the cell
free nucleic acids isolated from the plasma sample; measuring the
amount of the first mutation in the at least 2 serial dilutions
using digital droplet PCR; and evaluating linearity of the measured
dilutions to confirm accuracy of the method.
[0019] In some embodiments, the measuring of: (a) the first of the
pair of mutually exclusive mutations specific to the cancer in the
cell free nucleic acids isolated from the plasma sample obtained
from the subject and (b) the apparent amount of the first mutation
in cell free nucleic acids isolated from control plasma samples
obtained from control subjects known to have the second of the pair
of mutually exclusive mutations specific to the cancer is performed
by microarrays, Next-generation sequencing, chemiluminescence
methods, fluorescent methods, digital detection, and mass
spectrometry (MALDI-TOF).
[0020] Each of the limitations of the invention can encompass
various embodiments of the invention. It is, therefore, anticipated
that each of the limitations of the invention involving any one
element or combinations of elements can be included in each aspect
of the invention. This invention is not limited in its application
to the details of construction and the arrangement of components
set forth in the following description or illustrated in the
drawings. The invention is capable of other embodiments and of
being practiced or of being carried out in various ways.
[0021] Also, the phraseology and terminology used herein is for the
purpose of description and should not be regarded as limiting. The
use of "including," "comprising," or "having," "containing,"
"involving," and variations thereof herein, is meant to encompass
the items listed thereafter and equivalents thereof as well as
additional items.
[0022] These and other aspects of the inventions, as well as
various advantages and utilities will be apparent with reference to
the Detailed Description. Each aspect of the invention can
encompass various embodiments as will be understood.
[0023] All documents identified in this application are
incorporated in their entirety herein by reference.
BRIEF DESCRIPTION OF THE DRAWINGS
[0024] FIG. 1 shows the principle of digital droplet PCR as known
in the prior art. Digital droplet PCR (ddPCR) takes advantage of
recent developments in microfluids and surfactant chemistries. The
reaction mixture is divided into approximately 20000 droplets which
are PCR amplified, post-PCR fluorescently labeled and read in an
automated droplet flow cytometer. Each droplet is assigned a
positive and negative (1 or 0) value based on their fluorescent
intensity. The amount of positives and negatives are read by flow
cytometer and are used to calculate the concentration and the 95%
Poisson confidence levels.
[0025] FIGS. 2A-2C show the diagnostic accuracy of an embodiment of
the assays described herein. EGFR mutations were tested in plasma
from patients with KRAS-mutant NSCLC, genotype which is
non-overlapping. The low concentrations of EGFR mutations we
detected in this population can be considered the `normal range"
for analytical specificity (FIGS. 2A and 2B). Conversely, a KRAS
G12C assay was developed and the same specificity test was
performed (assaying for KRAS G12C mutation in EGFR and KRAS mutant
patients' plasma) (FIG. 2C).
[0026] FIG. 3A demonstrates the quality control platform developed
to optimize sensitivity of plasma DNA genotyping through monitoring
factors that impact DNA quantity, quality, and purity. Samples are
assayed for DNA quantity by measuring concentration of a
housekeeper gene (Line1). Line-1 amount greater 50,000 pg/uL
indicate sub optimal sample preparation and thereby impacting DNA
quantity, quality, and purity. Line-1 amount below a certain
threshold, in this case 50 pg/uL is indicative of too little input
material. FIG. 3B shows that the Line-1 DNA amount correlates to
total DNA amount in plasma.
[0027] FIGS. 4A-4B show preliminary data demonstrating that cfDNA
genotyping allows non-invasive monitoring of response in lung
cancer patients receiving therapy. In FIG. 4A the patient received
treatment, but continued to progress, whereas patient in FIG. 4B
received treatment and responded.
[0028] FIGS. 5A-5B demonstrate the monitoring evolution of
resistance mutations, in this case EGFR T790M. Patients with
EGFR-mutant lung cancer starting treatment with EGFR-targeted
therapy underwent serial monitoring of EGFR exon 19 and EGFR T790M
plasma genotype. Responding patients had normalization of their
plasma genotype. When resistance developed, the original EGFR
mutation again became detected (dashed line) as well as a new T790M
resistance mutation (solid line). Genotyping of the patient's tumor
at time of progression also demonstrated an acquired T790M
resistance mutation. Intriguingly, plasma T790M was detected 8
weeks prior to clinical progression. These findings suggest serial
cfDNA genotyping could allow monitoring for response as well as
assessment for new mutations when resistance develops (FIG. 5A).
The signal for acquired resistant (solid line in FIG. 5A) can be
used to guide treatment with second generation therapies
(demonstrated in FIG. 5B). In that case the resistance biomarker is
used to change treatment and after treatment it becomes a marker to
monitor whether the treatment works (similar to the dashed line in
FIG. 5A).
[0029] FIG. 6 shows more combinations of biomarkers.
[0030] FIGS. 7A-7D show the steps involved in digital droplet
PCR.
[0031] FIGS. 8A-8B show the EGFR del19 ddPCR assay.
[0032] FIGS. 9A-9F demonstrate the detection of mutant alleles in
gold standard positive and negative populations, using assays for
EGFR L858R (FIG. 9A), EGFR exon 19 deletion (FIG. 9B), and KRAS
G12C (FIG. 9C). Receiver operating curves are also shown (FIG. 9D,
9E, 9F). By studying plasma from lung cancer patients with a
non-overlapping genotype, a normal range for the EGFR assays is
identified to be 0-2 copies of L858R and 0-12 copies of exon 19
deletion per 100 .mu.L of cfDNA. Setting the threshold for positive
above this normal range, each assay has a sensitivity in the range
of 66-79% with 100% specificity.
[0033] FIGS. 10A-10B show plasma DNA quantification to optimize
sensitivity. (FIG. 10A demonstrates that a quantitative PCR for
LINE-1 can quantify cfDNA concentration and is highly correlated
with quantification using PicoGreen. Studying genotype
concentration in gold standard positive cases, the false negative
results all have either low or high levels of LINE-1 (FIG. 10 B).
Sensitivity is 100% when cfDNA concentration is optimal, with a
LINE-1 level between 3,000 and 650,000 pg/.mu.L (dashed lines).
Spheres represents EGFR-mutant cases and squares represents
KRAS-mutant cases.
[0034] FIGS. 11A-11D demonstrate serial measurement of plasma
genotype for disease monitoring. A wide dynamic range is seen in
some cases (FIG. 11A, 11B). Decreases in plasma genotype can be
seen both in cases of objective tumor shrinkage (FIG. 11A, 11D) and
in cases of symptomatic response with no measurable disease (FIG.
11B, 11C). Concurrent EGFR L858R (FIG. 11A, solid line) and T790M
(FIG. 11A, dashed line) mutations trend in parallel.
[0035] FIGS. 12A-12I show plasma levels of mutant EGFR in 9
patients (FIG. 12A-12I) receiving first-line erlotinib until
objective progression. In all patients, plasma levels of the EGFR
sensitizing mutation (solid line) drop in response to treatment,
with 8 patients (FIG. 12B-12I) having a complete plasma response.
In 6 patients, plasma genotype levels reemerge up to 4 months prior
to objective progression, and a lower concentration of T790M
(dashed line) is also detected. In 3 patients (FIG. 12G-12I),
plasma genotype was not detected at time of RECIST progression
(PD); all 3 had indolent progression in the chest only.
[0036] FIGS. 13A-13D show ddPCR assay characteristics. As the
sample input increases, the copies/.mu.L output increases in a
linear fashion across a wide dynamic range for both the L858R assay
(FIG. 13A) and the exon 19 deletion assay (FIG. 13B). Testing for
10 and 50 copies of mutant EGFR in a background of 1000 and 50,000
genome equivalents (GE), the L858R assay demonstrates more
consistent sensitivity (FIG. 13C) than the exon 19 deletion assay
(FIG. 13D).
[0037] FIG. 14 demonstrates detection of BRAF V600E in cfDNA from
patients with advanced melanoma. A threshold of 1 mutation/100
.mu.L DNA results in 86% sensitivity and 100% specificity.
[0038] FIGS. 15A-15B show inter- and intra-day variation of the
ddPCR assay. (FIG. 15A) Identical serial dilutions ranging from
10-10,000 T790M mutation copies per reaction were assayed in
triplicates on three nonconsecutive days. Percent coefficients of
variation ranged between 12.2-21.4% within days and 15.9-32.2%
between days. (FIG. 15B) Technical replicates of samples containing
either 1, 2, 10, or 20 copies of mutant T790M were assayed 32 times
on the same day. Results show that ddPCR exhibits
Poisson-distributed single molecule detection.
[0039] FIGS. 16A-16B show EGFR mutation concentration in NSCLC
patients. (FIG. 16A) Plasma genotype concentration is stable or
increases in patients without evidence of a response. (FIG. 16B) In
patients with at least a minor response to treatment.sup.1, plasma
genotype concentration.sup.2 decreases an average of 1773 fold.
.sup.1Minor response is defined as >10% reduction in tumor mass
on initial re-staging CT scan. .sup.2Includes both EGFR exon 19 del
and L858R depending on individual patient genotype. .sup.3A
threshold for detectable EGFR mutation was set as 0.5 copies/mL for
serial monitoring.
[0040] FIG. 17 shows a case report of a patient undergoing plasma
genotyping directed treatment
DETAILED DESCRIPTION OF THE INVENTION
[0041] The present application relates to the analysis and
monitoring of cell free DNA (cfDNA) for determining the
physiological state of an organism, to monitor drug efficacy and
dynamics, for early disease detection, as well as to ascertain
molecular markers and fingerprints of identified molecules in such
analysis to guide treatment. The methods of the invention provide
non-invasive blood-based quantitative assays to perform disease
diagnosis, monitoring, and pharmacodynamic assessment of drug
efficacy. The present invention has a number of advantages not
currently realized in clinical practice. First, the instant
invention allows serial sampling of each subject, i.e., successive
sampling of blood from the subject at different times. For example,
samples can be collected from the subject at different times during
therapy and/or before and after the subject has received any
therapy. Second, the instant invention enables a direct match
between a subject's tumor and therapeutic intervention, i.e, the
choice of anti-cancer therapies is guided by the tumor genotype.
Thirdly, it is broadly applicable across different cancer types.
The assays described herein are highly-specific (i.e., allow for
clinically actionable results by limiting false positives),
quantitative (i.e., have potential to be used to monitor response
to treatment) and are rapid (i.e., allow for a total turnaround
time (TAT) of 1-3 days).
[0042] The present invention is based on the finding that tumor
cells release cell free nucleic acids into body fluids, such as
blood. This tumor-related cell free DNA has diagnostic and
prognostic utility, and can be utilized for non-invasive tumor
genotyping, thereby eliminating the need for repeat tumor biopsies.
However, since these cell free nucleic acids are present in low
amounts in body fluids, it is difficult to accurately detect
genomic biomarkers in these nucleic acids as surrogates of tumor
diagnosis and progression, leading to a high percentage of false
positive and false negative results. In addition, procedures for
isolating cell free DNA from a body fluid may cause loss of the
cell free DNA and contamination by DNA released from cells present
in the body fluid. This usually results in a longer processing
time, a complicated processing method, a higher cost, and more
importantly, lower sensitivity, specificity, and consistency.
[0043] The inventors of the present invention have addressed these
problems by generating a control platform that allows an accurate
determination of whether a person carries the mutation of interest,
or whether the result obtained is an artifact of the measuring
assay. This platform is based on two concepts: (i) a quality
control step and (ii) a `gold standard` control population. The
quality control step identifies and utilizes a range of an amount
of a housekeeping gene and/or total DNA to confirm that the
isolated cell free nucleic acid is of sufficient quantity, quality
and/or purity, thereby ensuring that the sensitivity of described
methods. The `gold standard` control population is subjects with a
cancer having a mutation that does not exist in the test cancer
population. This population as a gold standard control group takes
into account two features. First, it recognizes that the blood of
cancer subjects can be modified relative to normal populations, and
therefore the control population is similar to the test population
in that respect. Second, it takes advantage of the fact that many
tumors exhibit mutually exclusive genetic mutations that are
non-overlapping in cancer subjects. Thus, for any given pair of
mutually exclusive mutations, there are test subjects who have (or
are suspected to have) a first of the pair of mutations and
"control subjects" that are known to have the second of the pair of
mutually exclusive mutations, but who, in fact, should have zero
amount of the first of the pair of mutually exclusive mutations
(because the first and second mutations do not co-occur). It was
discovered that these control subjects who only have the second
mutation can have background activity in assays that read as though
the first mutation also is present. The invention capitalizes on
this by making those subjects the control subjects. These control
subjects have a similar cancer and the `apparent` amount of the
first mutation measured in these control subjects represents the
"normal range" or "control amount". The control amount is believed
to be a very good measure of any artifacts or background
interference in the measuring assays.
[0044] According to some aspects of the invention, methods to
monitor cell free DNA (cfDNA) are provided. In some embodiments,
the term "cfDNA" is used interchangeably with "circulating DNA"
(ctDNA). The methods comprise obtaining a plasma sample from a
subject known to have a cancer characterized by a pair of mutually
exclusive mutations specific to the cancer; isolating cell free
nucleic acids from the plasma sample obtained from the subject;
measuring the amount a housekeeping gene and/or total DNA in the
cell free nucleic acids isolated from the plasma sample to confirm
that the amount of housekeeping gene and/or total DNA in the sample
is within a selected range; measuring the amount of a first of the
pair of mutually exclusive mutations specific to the cancer in the
cell free nucleic acids isolated from the plasma sample; and
indicating in a report that the subject has the first mutation when
(a) the amount of the housekeeping gene and/or total DNA in the
cell free nucleic acids isolated from the plasma sample is within
the selected range and (b) the amount of the first mutation is
increased as compared to a control amount, wherein the control
amount is determined by measuring the apparent amount of the first
mutation in control cell free nucleic acids isolated from plasma
samples obtained from control subjects known to have the second of
the pair of mutually exclusive mutations specific to the cancer
using measuring conditions substantially the same as those used to
measure the amount of the first mutation in the cell free nucleic
acids isolated from the plasma sample from the subject.
[0045] Cell free nucleic acids circulating in body fluids, such as
extra-cellular DNA fragments and mRNAs, are molecular biomarkers
for cancer. Unlike the uniformly truncated DNA released from
apoptotic cells, DNA released from cancer cells due to necrosis,
physical death, secretion, or disruption varies in size, and
displays tumor related characteristics, such as decreased strand
stability, oncogene and tumor suppressor gene mutations,
microsatellite alterations, and gene hypermethylation. The
detection of cancer-related mutations in the cell free nucleic
acids is clinically useful for the diagnosis and management of
cancer.
[0046] As used herein, "a pair of mutually exclusive mutations
specific to the cancer" means a pair of mutations that are
non-overlapping in cancer subjects. Many tumor profiling projects
have observed mutually exclusive genomic alterations across many
patients--for example, EGFR and KRAS are mutated in lung cancer,
but no patients harbor both genetic lesions. Additional
non-limiting examples in other cancer types include mutual
exclusivity between BRAF and KRAS mutations (both involved in the
common RAS/RAF signaling pathway) in colon cancer; BRAF and NRAS
mutations in melanoma; APC and CTNNB1 mutations (both involved in
the beta-catenin signaling pathway) in colorectal cancer, TP53
mutations and MDM2 copy number amplification in glioblastomas and
mutual exclusivity between BRCA1/2 mutations and BRCA1 epigenetic
silencing in serous ovarian cancer (The Cancer Genome Atlas
Research Network 2011; Ciriello et al, Genome Research 2011; The
Cancer Genome Atlas Research Network 2008). Other examples of
mutually exclusive mutations are described in Cui Q, PLoS One.
2010).
[0047] A cancer characterized by a pair of mutually exclusive
mutations specific to the cancer is a cancer that has a pair of
mutually exclusive mutations. In some embodiments, these mutations
are "passenger" mutations, i.e., they are functionally neutral and
do not contribute to tumor development. In preferred embodiments,
these mutations are "driver" mutations, i.e., they contribute to
the tumorigenesis. Non-limiting examples of cancer include lung
cancer, colon cancer, melanoma, ovarian cancer, breast cancer,
glioblastomas, thyroid cancer, and prostate cancer.
[0048] In some embodiments, the cancer is lung cancer, and the pair
of mutually exclusive mutations comprises an epidermal growth
factor receptor (EGFR) mutation and a v-Ki-ras2 Kirsten rat sarcoma
viral oncogene homolog (KRAS) mutation. In some embodiments, the
EGFR mutation is selected from the group consisting of: leucine (L)
to an arginine (R) substitution at position 858 (L858R), threonine
(T) to a methionine (M) substitution at position 790 (T790M),
leucine (L) to a glutamine (Q) substitution at position 861
(L861Q), glycine (G) to a serine (S) substitution at position 719
(G719S), exon 19 deletions (del 19) and exon 20 insertions. In some
embodiments, the KRAS mutation is glycine (G) to a cysteine (C)
substitution at position 12 (G12C).
[0049] In some embodiments, the cancer is colon cancer, and the
pair of mutually exclusive mutations comprises a v-raf murine
sarcoma viral oncogene homolog B1 (BRAF) mutation and a v-Ki-ras2
Kirsten rat sarcoma viral oncogene homolog (KRAS) mutation. In some
embodiments, the BRAF mutation is a valine (V) to a glutamic acid
(E) substitution at position 600 (V600E).
[0050] In some embodiments, the cancer is a melanoma, and the pair
of mutually exclusive mutations comprises a v-raf murine sarcoma
viral oncogene homolog B1 (BRAF) mutation and a neuroblastoma RAS
viral (v-ras) oncogene homolog (NRAS) mutation.
[0051] "Subject" as used herein, refers to a human or animal,
including all vertebrates, e.g., mammals such as primates
(particularly higher primates), sheep, dog, rodents (e.g., mouse or
rat), guinea pig, goat, pig, cat, rabbit, and cow, etc. Typically,
the subject is a human, and is diagnosed with cancer using any
suitable diagnostic method known in the art. For example a subject
may be diagnosed with cancer using one or more of the following
techniques: histopathology, imaging tests, and blood tests. Once
the subject has been diagnosed with cancer, the type of cancer will
determine whether the present invention can be used to monitor cell
free nucleic acids. Thus, an additional determination is made
whether the cancer characterized by a pair of mutually exclusive
mutations specific to the cancer, i.e., whether the subject has a
genetic mutation of a pair of mutually exclusive mutations specific
to the cancer. The presence of the mutation can be determined using
any suitable diagnostic method known in the art, for example, by
tumor genotyping.
[0052] In some embodiments, any body fluid sample containing cell
free DNA released by cancer cells can be used in the methods
described herein. Examples of such body fluids include, without
limitation, blood (serum/plasma), bone marrow (serum/plasma),
cerebral spinal fluid, peritoneal fluid, pleural fluid, lymph
fluid, ascites, serous fluid, sputum, lacrimal fluid, stool, urine,
saliva, ductal fluid from breast, gastric juice, and pancreatic
juice. In some embodiments, the sample used is blood. In preferred
embodiments, the sample used is serum or plasma. In some preferred
embodiments, the sample used is plasma. For cell free DNA in
plasma, the concentration can range from 1-100 ng/ml in human
samples.
[0053] Body fluids can be collected using any of the standard
methods known in the art. Obtaining a plasma sample from a subject
means taking possession of a plasma sample of the subject. In some
embodiments, the plasma sample may be removed from the subject by a
medical practitioner (e.g., a doctor, nurse, or a clinical
laboratory practitioner), and then provided to the person
performing the measuring steps of the assay described herein. The
plasma sample may be provided to the person performing the
measuring steps by the subject or by a medical practitioner (e.g.,
a doctor, nurse, or a clinical laboratory practitioner). In some
embodiments, the person performing the measuring steps obtains a
plasma sample from the subject by removing a blood sample from the
subject and isolating plasma from the blood sample.
[0054] Cell free DNA from a biological/plasma sample can be
isolated from the bodily fluid/plasma samples using any method
known in the art. For example, the potentially contaminating cells
can be removed from a body fluid by centrifugation and/or
filtration. The proteins that may interfere with the detection of
the cell free DNA can be removed, e.g., by proteinase K digestion.
The cell free DNA may be further purified after removal of the
cells and proteins from the body fluid, using any of the methods
known in the art. For example, the cell free DNA may be extracted
with phenol, precipitated in alcohol, and dissolved in an aqueous
solution.
[0055] Isolation of cell free DNA from a body fluid may cause loss
of the DNA and contamination by DNA released from cells present in
the body fluid. This usually results in a longer processing time, a
complicated processing method, a higher cost, and lower
sensitivity, specificity, and consistency. The inventors of the
present invention have developed a quality control platform to
optimize the calling criteria of the cell free tumor DNA assay
described herein. Thus, as a quality control step, the methods
described herein utilize the amount of a housekeeping gene and/or
total DNA to confirm that the isolated cell free nucleic acid is of
sufficient quantity, quality and/or purity so as to ensure that the
sensitivity of described methods is accurate. Housekeeping genes
are typically constitutive genes that are required for the
maintenance of basic cellular function, and are expressed in all
cells of an organism under normal and pathophysiological
conditions. Non-limiting examples of housekeeping genes include
Line1, GAPDH, HSP90, .beta.-actin, and .beta.-2-microglobulin.
Samples are assayed for quality by measuring the amount of a
housekeeping gene and/or total DNA in the cell free nucleic acids
isolated from the plasma sample, and confirming that the amount of
the housekeeping gene and/or total DNA in the sample is within a
selected range. An amount of the housekeeping gene and/or total DNA
higher than the selected range indicates suboptimal sample
preparation and blood lysis which impacts DNA quantity, quality
and/or purity. An amount lower than the selected range is
indicative of too little input material. One of ordinary skill in
the art can determine the "selected range" using methods known in
the art. In some embodiments, the housekeeping gene is Line1 and
the selected range is between 100,000 pg/.mu.l and 10 pg/.mu.l. In
some embodiments, the housekeeping gene is Line1, and the selected
range is between 75,000 pg/.mu.l and 25 pg/.mu.l. In preferred
embodiments, the housekeeping gene is Line1 and the selected range
is between 50,000 pg/.mu.l and 50 pg/.mu.l. This quality control
step can be performed before, after or simultaneously with the
other measuring steps of the methods described herein.
[0056] The amount of the (i) housekeeping gene and/or total DNA,
and (ii) the first mutation in the cell free nucleic acids isolated
from the plasma sample can be determined using a number of methods
well known in the art, e.g., quantitative PCR(qPCR), microarrays,
Next-generation sequencing, or gel electrophoresis based,
colorimetric detection assays such as chemiluminescence methods,
fluorescent methods, digital detection, and mass spectrometry
(e.g., MALDI-TOF). In a preferred embodiment, qPCR is employed
since it allows routine and reliable quantification of PCR
products. In some preferred embodiments, digital droplet PCR is
used to determine the amount of the (i) housekeeping gene and/or
total DNA, and (ii) the first mutation in the cell free nucleic
acids isolated from the plasma sample. The fundamental advantages
that digital droplet PCR (ddPCR) offers are (a) an increase in
dynamic range, (b) improvement in precision of detecting small
changes in template DNA, (c) its ability to tolerate a wide range
of amplification efficiencies, and (d) its ability to measure
absolute DNA concentrations.
[0057] A "control amount" is determined by measuring the apparent
amount of the first mutation in control cell free nucleic acids
isolated from plasma samples obtained from control subjects known
to have the second of the pair of mutually exclusive mutations
specific to the cancer. The control amount is measured under
conditions that are substantially the same as those used to measure
the amount of the first mutation in the cell free nucleic acids
isolated from the plasma sample from the subject. Since the pair of
mutually exclusive mutations are non-overlapping in cancer
subjects, the amount of the first mutation in control cell free
nucleic acids obtained from control subjects known to have the
second of the pair of mutually exclusive mutations specific to the
cancer is expected to be zero (because the first and second
mutations do not co-occur). However, the quantification assay and
the measuring conditions used may lead to the detection of an
apparent or superficial amount of the first mutation in subjects
known to have the second mutation. Thus, these control subjects who
only have the second mutation can have background activity in
assays that read as though the first mutation also is present.
These control subjects have a similar cancer and the `apparent`
amount of the first mutation measured in these control subjects
represents the "normal range" or "control amount". The control
amount is believed to be a very good measure of any artifacts or
background interference in the measuring assays. For example, the
amount of EGFR mutation in cell free DNA in plasma samples from
subjects with KRAS-mutant non-small cell lung cancer is expected to
be zero, since EGFR mutations and the KRAS mutations are
non-overlapping in lung cancer. However, presence of the EGFR
mutation was detected in a very low amount in subjects with
KRAS-mutant lung cancer, indicating that this is the "normal range"
for specificity (FIGS. 2A-2C), which represents an artifact or
background interference in the measuring assay. In some
embodiments, the control amount for the L858R and del 19 mutations
from KRAS mutant cancer is 0-10 and 0-1 copies/ml.
[0058] A tangible or electronic report indicating the results of
the analysis, i.e. the subject has the first mutation when (a) the
amount of the housekeeping gene and/or total DNA in the cell free
nucleic acids isolated from the plasma sample is within the
selected range and (b) the amount of the first mutation is
increased as compared to a control amount, and any other
information pertaining to the analysis could optionally be
generated as part of the analysis (which may be interchangeably
referred to herein as "providing" a report, "producing" a report,
or "generating" a report). Examples of reports may include, but are
not limited to, reports in paper (such as computer-generated
printouts of test results) or equivalent formats and reports stored
on computer readable medium (such as a CD, computer hard drive, or
computer network server, etc.). Reports, particularly those stored
on computer readable medium, can be part of a database (such as a
database of patient records, which may be a "secure database" that
has security features that limit access to the report, such as to
allow only the patient and the patient's medical practitioners to
view the report, for example).
[0059] A report can further be transmitted, communicated or
reported (these terms may be used herein interchangeably), such as
to the subject who was tested, a medical practitioner (e.g., a
doctor, nurse, clinical laboratory practitioner, genetic counselor,
etc.), a healthcare organization, a clinical laboratory, and/or any
other party intended to view or possess the report. The act of
`transmitting` or `communicating` a report can be by any means
known in the art, based on the form of the report, and includes
both oral and non-oral transmission. Furthermore, "transmitting" or
"communicating" a report can include delivering a report
("pushing") and/or retrieving ("pulling") a report. For example,
reports can be transmitted/communicated by such means as being
physically transferred between parties (such as for reports in
paper format), such as by being physically delivered from one party
to another, or by being transmitted electronically or in signal
form (e.g., via e-mail or over the internet, by facsimile, and/or
by any wired or wireless communication methods known in the art),
such as by being retrieved from a database stored on a computer
network server, etc.
[0060] In some embodiments, the amount of the (i) housekeeping gene
and/or total DNA, and (ii) the first of the pair of mutually
exclusive mutations specific to the cancer is determined by
preparing at least 2 serial dilutions of the cell free nucleic
acids isolated from the plasma sample; measuring the amount of the
(i) housekeeping gene and/or total DNA, and (ii) the first mutation
in the at least 2 serial dilutions using digital droplet PCR; and
evaluating linearity of the measured dilutions to confirm accuracy
of the method. Linearity of dilution refers to the ability of the
analytical method, within the assay range to obtain test results
that are close to the expected amount of the mutation in the
diluted sample. Linearity is measured by the r-squared (r.sup.2
coefficient of determination, or r, coefficient of correlation)
value for the linear regression of the expected versus observed
concentration.
[0061] In some embodiments, the amount of the first mutation is
measured before and after administration of a an anti-cancer
therapy to the subject. As used herein, "anti-cancer therapy"
refers to any therapy that has as a goal to reduce the severity of
a cancer or to at least partially eliminate a cancer.
Alternatively, "anti-cancer therapy" refers to any therapy that has
as a goal to reduce or to at least partially eliminate metastasis
of a cancer. Anti-cancer therapy includes chemotherapy, radiation,
surgery, and some combination of these and other therapeutic
options. In some embodiments, therapy targeted to the first of the
pair of mutually exclusive mutations specific to the cancer is
administered to the subject.
[0062] In some embodiments, the amount of the housekeeping gene
and/or total DNA in the cell free nucleic acids isolated from the
plasma sample and (b) the amount of the first mutation is measured
repeatedly so as to monitor the subject's amount of the first
mutation over time. In some embodiments, the amount of the first
mutation is measured in a first sample that is obtained from the
subject before the subject has received any anti-cancer therapy,
and in a second sample that is obtained from the subject after the
subject has received an anti-cancer therapy. In some embodiments, a
decrease in amount of the first mutation over time indicates that
the cancer is stabilizing or decreasing. In some embodiments, an
increase in amount of the first mutation over time indicates that
the cancer is increasing.
[0063] According to some aspects of the invention, a method to
monitor efficacy of anti-cancer therapy is provided. The method
comprises administering an anti-cancer therapy to a subject known
to have a cancer characterized by a pair of mutually exclusive
mutations specific to the cancer; obtaining a plasma sample from
the subject; isolating cell free nucleic acids from the plasma
sample obtained from the subject; measuring the amount a
housekeeping gene and/or total DNA in the cell free nucleic acids
isolated from the plasma sample to confirm that the amount of
housekeeping gene and/or total DNA in the sample is within a
selected range; measuring the amount of a first of the pair of
mutually exclusive mutations specific to the cancer in the cell
free nucleic acids isolated from the plasma sample; and measuring
the apparent amount of the first mutation in control cell free
nucleic acids isolated from plasma samples obtained from control
subjects known to have the second of the pair of mutually exclusive
mutations specific to the cancer using measuring conditions
substantially the same as those used to measure the amount of the
first mutation in the cell free nucleic acids isolated from the
plasma sample from the subject.
[0064] In some embodiments, the amount of the first mutation is
measured before and after administration of the anti-cancer therapy
to the subject. In some embodiments, the measuring steps are
repeated so as to monitor the subject's amount of the first
mutation over time. The anti-cancer therapy is considered to be
efficacious, i.e., successful in producing the desired result, when
the amount of the mutation decreases over time. The anti-cancer
therapy is not efficacious, i.e., not successful in producing the
desired result, when the amount of the mutation increases over
time. In some embodiments, the subject's amount of the first
mutation is measured: (a) in a first sample obtained from the
subject before the subject received the anti-cancer therapy; and
(b) in a second sample obtained from the subject after the subject
received the anti-cancer therapy.
[0065] According to some aspects of the invention, a method to
treat cancer is provided. The method comprises obtaining a plasma
sample from a subject known to have a cancer characterized by a
pair of mutually exclusive mutations specific to the cancer;
isolating cell free nucleic acids from the plasma sample obtained
from the subject; measuring the amount a housekeeping gene and/or
total DNA in the cell free nucleic acids isolated from the plasma
sample to confirm that the amount of housekeeping gene and/or total
DNA in the sample is within a selected range; measuring the amount
of a first of the pair of mutually exclusive mutations specific to
the cancer in the cell free nucleic acids isolated from the plasma
sample; measuring the apparent amount of the first mutation in
control cell free nucleic acids isolated from plasma samples
obtained from control subjects known to have the second of the pair
of mutually exclusive mutations specific to the cancer using
measuring conditions substantially the same as those used to
measure the amount of the first mutation in the cell free nucleic
acids isolated from the plasma sample from the subject; and
treating the subject with an anti-cancer therapy when (a) the
amount of the housekeeping gene and/or total DNA in the cell free
nucleic acids isolated from the plasma sample is within the
selected range and (b) the amount of the first mutation is
increased as compared to a control amount.
[0066] The subject can be treated with an effective amount of any
anti-cancer therapy. In some embodiments, the amount of the first
mutation is measured before and after administration of the
anti-cancer therapy to the subject. In some embodiments, the
measuring steps are repeated so as to monitor the subject's amount
of the first mutation over time. Administration of the anti-cancer
therapy is maintained when the amount of the mutation decreases
over time.
[0067] Alternatively, the anti-cancer therapy is administered at a
higher dosage or is changed when the amount of the mutation
increases over time and/or a new mutation known to confer drug
resistance (e.g., T790M) is measured.
[0068] The present invention is further illustrated by the
following Examples, which in no way should be construed as further
limiting. The entire contents of all of the references (including
literature references, issued patents, published patent
applications, and co pending patent applications) cited throughout
this application are hereby expressly incorporated by
reference.
EXAMPLES
Example 1: Protocols for Sample Preparation and Droplet Digital PCR
(ddPCR)
[0069] Plasma Isolation from Whole Blood
A. Equipment and Reagents
BD EDTA Tubes--Glass (BD #366450)
[0070] 5-15 ml polypropylene tube
Pipettor.about.1000 .mu.l,
[0071] RNase/DNase-free pipet tips (aerosol barrier)--1000 .mu.l 15
ml polypropylene centrifuge tubes
Tabletop Centrifuge
B. Procedure
[0072] *To optimize DNA yield, about 10 ml of whole blood are
required for each specimen. **Plasma isolation should be carried
out within one (1) hour of blood draw. 1. Remix the blood sample
immediately prior to centrifugation. 2. Centrifuge the EDTA tubes
at room temperature in a horizontal rotor (swing-out head) for 10
minutes at 1900 g (3000 rpm). 3. Without disturbing the whitish
layer of mononuclear cells and platelets, aspirate the plasma using
a micropipette and transfer to a 15 ml polypropylene conical tube.
4. Centrifuge the conical tube at 1900 g (3000 rpm) for 10 minutes
at 40 C. Carefully transfer the plasma to a fresh 5-15 ml
polypropylene tube, leave about 0.5 ml at the bottom of the tube
undisturbed. About 4-5 ml of plasma can be obtained from 10 ml of
whole blood sample. Polystylene tubes should not be used for this
purpose. They will crack in -80.degree. C. freezer. 5. Proceed to
cfDNA extraction or immediately store the isolated plasma at
-80.degree. C. Thaw plasma samples at room temperature on the day
of use.
Cell-Free DNA Extraction
A. Background
[0073] Cell-free nucleic acids, such as tumor-specific
extracellular DNA fragments and mRNAs in the blood or fetal nucleic
acids in maternal blood, are present in serum or plasma usually as
short fragments, <1000 bp (DNA) or <1000 nt (RNA). In
addition, cell free miRNAs, as small as 20 nt, have the potential
to provide biomarkers for certain cancers and disease states. The
concentration of cell free nucleic acids in biological fluids such
as plasma, serum, or urine, is usually low and varies considerably
among different individuals. For cell free DNA in plasma, the
concentration can range from 1-100 ng/ml in human samples. In
samples obtained from different individuals, a similar
sample-to-sample variability can be assumed for the concentration
of cell free messenger RNA fragments and miRNA molecules.
B. Equipment and Reagents
Microfuge Centrifuge
SterilGARD Hood
[0074] Water bath or heating block capable of holding 50 ml
centrifuge tubes at 60.degree. C. Heating block or similar at
56.degree. C. (capable of holding 2 ml collection tubes)
Daigger Vortex Genie 2
Pipettors--20 .mu.l, 200 .mu.l, 1000 .mu.l,
[0075] RNase/DNase-free pipet tips (aerosol barrier) --20 .mu.l,
200 .mu.l, 1000 .mu.l 1.5 ml microcentrifuge tubes (Fisher
#02-681-461) 50 ml centrifuge tubes
100% Ethanol
100% Isopropanol
[0076] Phosphate-buffered saline (PBS)
QIAamp Circulating Nucleic Acid Kit (Qiagen #55114)
Crushed Ice
C. Protocol
[0077] Before starting, make sure that buffers are prepared
according to specifications in Qiagen QIAamp Circulating Nucleic
Acids Kit manual. Wipe down lab bench, hood and pipetters with 70%
ethanol.
Buffer ACB*
[0078] Before use, add 200 ml isopropanol (100%) to 300 ml buffer
ACB concentrate to obtain 500 ml Buffer ACB. Mix well after adding
isopropanol.
Buffer ACW1*
[0079] Before use, add 25 ml ethanol (96-100%) to 19 ml buffer ACW1
concentrate to obtain 44 ml Buffer ACW1. Mix well after adding
ethanol.
Buffer ACW2.dagger.
[0080] Before use, add 30 ml ethanol (96-100%) to 13 ml buffer ACW2
concentrate to obtain 43 ml Buffer ACW2. Mix well after adding
ethanol. Adding carrier RNA to Buffer ACL* Carrier RNA serves two
purposes. Firstly, it enhances binding of nucleic acids to the
QIAamp Mini membrane, especially if there are very few target
molecules in the sample. Secondly, the addition of large amounts of
carrier RNA reduces the chance of RNA degradation in the rare event
that RNase molecules escape denaturation by the chaotropic salts
and detergent in Buffer ACL. Add 1550 .mu.l Buffer AVE to the tube
containing 310 g lyophilized carrier RNA to obtain a solution of
0.2 .mu.g/.mu.l. Dissolve the carrier RNA thoroughly, divide it
into conveniently sized aliquots, and store it at -15 to
-30.degree. C. Do not freeze-thaw the aliquots of carrier RNA more
than three times. Note that carrier RNA does not dissolve in Buffer
ACL. It must first be dissolved in Buffer AVE and then added to
Buffer ACL. Calculate the volume of Buffer ACL-carrier RNA mix
needed per batch of samples according to the tables in the kit
manual. Select the number of samples to be simultaneously
processed. Gently mix by inverting the tube or bottle 10 times. To
avoid foaming, do not vortex. Protocol: Purification of cell free
Nucleic Acids from 4 ml or 5 ml Serum or Plasma For 1 ml, 2 ml, or
3 ml, see Qiagen kit manual, page 22.
Important Points Before Starting
[0081] All centrifugation steps are carried out at room temperature
(15-25.degree. C.). Switch off vacuum between steps to ensure that
a consistent, even vacuum is applied during protocol steps.
Things to do Before Starting
[0082] Equilibrate samples to room temperature. If samples are
<4 ml or <5 ml, bring the volumes up to 4 ml or 5 ml with
phosphate-buffered saline.
Set up the QIAvac 24 Plus.
[0083] Heat a water bath or heating block to 60.degree. C. for use
with 50 ml centrifuge tubes in step 4. Heat a heating block to
56.degree. C. for use with 2 ml collection tubes in step 14.
Equilibrate Buffer AVE to room temperature for elution in step 15.
Ensure that Buffer ACB, Buffer ACW1, and Buffer ACW2 have been
prepared. Add carrier RNA reconstituted in Buffer AVE to Buffer ACL
according to instructions in the table below.
TABLE-US-00001 TABLE 1 Volumes of Buffer ACL and carrier RNA
(dissolved in Buffer AVE) required for processing .tangle-solidup.
4 ml and .circle-solid. 5 ml samples Buffer ACL (ml) Number of
Carrier RNA in samples .tangle-solidup. .circle-solid. Buffer AVE
(.mu.l) 1 3.5 4.4 5.6 2 7.0 8.8 11.3 3 10.6 13.2 16.9 4 14.1 17.6
22.5 5 17.6 22.0 28.1 6 21.1 26.4 33.8 7 24.6 30.8 39.4 8 28.2 35.2
45.0 9 31.7 39.6 50.6 10 35.2 44.0 56.3 11 38.7 48.4 61.9 12 42.2
52.8 67.5 13 45.8 57.2 73.1 14 49.3 61.6 78.8 15 52.8 66.0 84.4 16
56.3 70.4 90.0 17 59.8 74.8 95.6 18 63.4 79.2 101.3 19 66.9 83.6
106.9 20 70.4 88.0 112.5 21 73.9 92.4 118.1 22 77.4 96.8 123.8 23
81.0 101.2 129.4 24 84.5 105.6 135.0
Procedure
[0084] 1. Pipet 400 .mu.l or 500 .mu.l QIAGEN Proteinase K into a
50 ml centrifuge tube. 2. Add 4 ml or 5 ml of serum or plasma to
the tube. 3. Add 3.2 ml or 4.0 ml Buffer ACL (containing 1.0 g
carrier RNA). Close the cap and mix by pulse-vortexing for 30 s.
Make sure that a visible vortex forms in the tube. To ensure
efficient lysis, it is essential that the sample and Buffer ACL are
mixed thoroughly to yield a homogeneous solution. Note: Do not
interrupt the procedure at this time. Proceed immediately to step 4
to start the lysis incubation.
4. Incubate at 60.degree. C. for 30 min.
[0085] 5. Place the tube back on the lab bench and unscrew the cap.
6. Add 7.2 ml or 9 ml Buffer ACB to the lysate in the tube. Close
the cap and mix thoroughly by pulse-vortexing for 15-30 s. 7.
Incubate the lysate-Buffer ACB mixture in the tube for 5 min on
ice. 8. 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.
Note: Keep the collection tube for the dry spin in step 13. 9.
Carefully apply the lysate-Buffer ACB mixture from step 7 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 20 ml when starting with 5 ml
sample) may need up to 15 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). Note: To avoid cross-contamination, be
careful not to move the tube extenders over neighboring QIAamp Mini
Columns. 10. 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. 11. 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. 12. 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. 13.
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. 14. 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. 15. 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. Important: 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. 16.
Centrifuge in a microcentrifuge at full speed (20,000.times.g;
14,000 rpm) for 1 min to elute the nucleic acids. D. Storage--DNA
shall be stored in 1.5 ml eppendorf tubes at 4.degree. C. for
immediate use. DNA shall be stored at -80.degree. C.
indefinitely.
E. Troubleshooting
[0086] Little or No Nucleic Acids in the Eluate
b) Extended time between blood draw and plasma preparation. Cells
may disintegrate and release genomic DNA into the plasma, diluting
the target nucleic acid. e) Buffers not prepared correctly.
[0087] General Handling
Clogged QIAamp Mini Column
[0088] Place the QIAamp Mini column in a 2 ml collection tube and
spin it at full speed for 1 minute or until sample has completely
passed through the membrane. Re-assemble the QIAamp Mini column
with Tube Extender, VacConnector and (optional) VacValve. Transfer
the remaining sample lysate into the Tube Extender, switch on the
vacuum pump, open the VacValve, and pass the remaining lysate
through the QIAamp Mini column. Repeat the above procedure if the
QIAamp Mini column continues to clog.
[0089] Cryoprecipitates may have formed in plasma due to repeated
freezing and thawing. These can block the QIAamp Mini column. Do
not use plasma that has been frozen and thawed more than once. In
case cryoprecipitates are visible clear the sample by
centrifugation for 5 min at 16,000 g.
Droplet Digital PCR
A. Background
[0090] Droplet Digital.TM. PCR (ddPCR.TM.) provides an absolute
quantitation of target DNA molecules with accuracy, precision, and
sensitivity. ddPCR applications include measurement of copy number
variation, rare sequence detection, mutation detection, and gene
expression analysis (FIGS. 7A-7D).
B. Equipment and Reagents
Bio-Rad Tetra-Head or My-iQ Thermal Cycler
[0091] QX100 ddPCR system
Eppendorf PCR Plate Heat Sealer
Pipettors--2, 20, 200, 1000 ul
[0092] RNase/DNase-free pipet tips (aerosol barrier) --20 .mu.l,
200 .mu.l, 1000 .mu.l 1.5 ml microcentrifuge tubes Dnase-free,
Rnase-free water Twin Tec semi-skirted 96-well plates (Eppendorf
#951020362)
Easy Pierce Foil PCR Plate Seals (Thermo-Fisher #AB-0757)
[0093] Droplet reader oil (Bio-Rad #1863004) Droplet generation oil
(Bio-Rad #1863005) DG8 cartridge for ddPCR (Bio-Rad #1863008) DG8
caskets for ddPCR (Bio-Rad #1863009) 2.times.ddPCR supermix for
probes (Bio-Rad #1863010) 40.times.Tagman primer/probe mix (Life
Technologies)
C. Precautions
[0094] As a general rule, set up the laboratory to avoid
contamination: Wipe down work surfaces using 70% ethanol: hood,
bench, racks, pipettes, cartridge holders, waste beaker, droplet
generator and heat sealer before you start and after you finish.
Put UV (15 min timer) on when you are done in the hood (or ask
another clean room user to do that for you when she finishes after
you.) Clean the hood on weekly basis using DNA Zap. Change gloves
frequently: always use CLEAN gloves when prepare master mix,
especially when open a Tagman probe tube. Change gloves between
handling positive controls and patient samples. Use aerosol
resistant pipette tips and calibrated pipettes. Check liquid level
in the tip before/after pipetting. Pipette into each reaction
vessel once. Have your own set of PCR reagents. Store the reagents
(including water) in small aliquots.
D. Protocols
[0095] i. Preparation of ddPCR Reactions: * Remember to include a
no template (water), wildtype and mutant control for every master
mix. 1. When running multiple reactions, always make a master mix
(with 10% extra volume) without the template. Add components in the
following order, mix up and down several times by pipetting.
TABLE-US-00002 Final Reagent concentration Water * 2xddPCR Supermix
for Probes 1x 40xTaqman primer/probe mix 1x
2. Aliquot into the sample wells of the cartridges and add the DNA
samples last. It is important to fill sample wells before filling
oil wells (70 ul of Droplet Generation Oil) of the DG8 cartridges.
3. Cover the cartridges with a piece of DG8 gasket and load the
cartridge into Droplet Generator. 4. When the light on Droplet
Generator turns green, take out the cartridge. 5. Use a manual 50
ul 8-well channel pipette, gently pipette up 30 ul droplets while
counting to five. Release the droplets into a 96-well PCR plate
while counting to five. 6. Repeat droplet generation until all the
cartridges are processed. 7. Cover the PCR plate with a sheet of
Easy Pierce Foil PCR Plate Seal. Mark well A1 at the right corner.
Seal the PCR plate with pre-heated Eppendorf PCR Plate Sealer:
press down hard (second tier) and count six times. Flip the plate
and press hard, count to six. Remember to turn off the plate sealer
after you are done. 8. Place the plate in a thermal cycler with
pre-set ddPCR programs. 9. Select the appropriate program and start
the PCR. ii. Plate Reading on QX100 Reader: 1. Twenty minutes
before the PCR program finishes, set up a new plate layout in
QuantaSoft program. 2. Check and make sure the lights indicating
levels of the QX100 reader oil and waste are green. 3. Prime the
QX100 reader. 4. Transfer the finished PCR plate to the QX100
reader and begin reading. 5. Shut down the QX100 reader and
instrument-attached laptop every Friday afternoon. iii. ddPCR
Cycling Conditions: These programs were developed for Bio-Rad
Tetra-Head and My-iQ cyclers. Other thermocyclers may require
different profiles.
TABLE-US-00003 Step Temperature 1 95 C. 10 min Ramp to 94 C. 2.5
C./sec 2 94 C. 30 sec Ramp to annealing temp 2.5/sec 3 Annealing
temp* 1 min 2 and 3 40 cycles 4 10 C. hold *See next section for
annealing temperatures specific for each mutation detection
assay.
iv. ddPCR Programs and Controls by Gene/Exon:
TABLE-US-00004 Gene/ PCR Mutation program Positive Controls EGFR
Del 19 ddPCR_55 PC9 (del19) and A549 (EGFRwt) gDNA L858R ddPCR_58
plasmids T790M ddPCR_58 plasmids KRAS G12C ddPCR_60 plasmids G12D
ddPCR_61 plasmids G12S ddPCR_64 A549 (G12S) and PC9 (KRASwt)
gDNA
E. Custom Designed EGFR Del19 ddPCR Assay i. Assay Background:
Various exon 19 deletions are the most common EGFR activating
mutations in NSCLC patients. This particular EGFR del 19 ddPCR
assay is used to detect a deletion within exon 19 that causes a 4-5
amino acid deletion within the kinase domain of EGFR. Due to the
short length of the exon (99 bp), the design of the primers extends
to intronic sequences. Of the two primers, the forward primer lies
in the exonic sequence, the reverse primer lies in the intronic
sequence between exons 19 and 20. Of the two probes, the
VIC-labeled "reference probe" sequence is shared by both the
wildtype and the deletion mutants; the FAM-labeled probe sequence
spans the hotspots of deletion area and is only present in EGFR
ex19 wildtype samples. An EGFR ex19 wildtype sample will have both
FAM- and VIC-labeled droplets, while an EGFR del19 mutant sample
will only have VIC-labeled droplets (FIGS. 8A-8B). The two
populations can be easily grouped by free-hand function in
QuantaSoft software.
TABLE-US-00005 Forward Primer: GTGAGAAAGTTAAAATTCCCGTC (SEQ ID NO:
1) 39% GC 58.4.degree. Tm Reverse Primer: CACACAGCAAAGCAGAAAC (SEQ
ID NO: 2) 47% GC 58.7.degree. Tm Probe 1 (wildtype-specific)
5'-FAM-AGGAATTAAGAGAAGCAACATC-MGB-3' (SEQ ID NO: 3) 36% GC
72.2.degree. Tm Probe 2 (reference)
5'-VIC-ATCGAGGATTTCCTTGTTG-MGB-3' (SEQ ID NO: 4) 42% GC
68.8.degree. Tm
Results
[0096] Digital droplet PCR was used to develop a method of
assessing tumor derived DNA from plasma samples of cancer patients.
Digital droplet PCR (ddPCR) takes advantage of recent developments
in microfluids and surfactant chemistries. The reaction mixture is
divided into approximately 20000 droplets which are PCR amplified,
post-PCR fluorescently labeled and read in an automated droplet
flow cytometer. Each droplet is assigned a positive and negative (1
or 0) value based on their fluorescent intensity. The amount of
positives and negatives are read by flow cytometer and are used to
calculate the concentration and the 95% Poisson confidence levels
(FIG. 1). The fundamental advantages that digital droplet PCR
(ddPCR) offers are many, including (a) an increase in dynamic
range, (b) improvement in precision of detecting small changes in
template DNA, (c) its ability to tolerate a wide range of
amplification efficiencies, and (d) its ability to measure absolute
DNA concentrations.
[0097] A particular challenge with high sensitivity assays is
identifying a "gold standard" wild-type population given that
conventional tumor genotyping does have a chance of being falsely
negative, some wild-type cancers may actually carry the genotype of
interest. To overcome this challenge, EGFR mutations were tested in
plasma from patients with KRAS-mutant NSCLC, genotype which is
non-overlapping. Thus, the low concentrations of EGFR mutations
detected in this population can be considered the "normal range"
for analytical specificity (FIGS. 2A and B). Conversely, a KRAS
G12C assay was developed and the same specificity test was
performed (assaying for KRAS G12C mutation in EGFR and KRAS mut
patients' plasma) (FIG. 2C).
[0098] A quality control platform was developed to optimize the
calling criteria of our ctDNA assay (]FIG. 3A). Samples are assayed
for DNA quantity by measuring concentration of a housekeeper gene
(Line1). Line-1 concentration greater than 50,000 pg/uL indicate
sub optimal sample preparation and thereby impacting DNA quantity,
quality, and purity. Line-1 concentrations below a certain
threshold, in this case 50 pg/uL is indicative of too little input
material. Line-1 DNA concentration correlates to total DNA
concentration in plasma (FIG. 3B). Thus both Line-1 and/or total
DNA concentration could be used for quality control.
[0099] Preliminary data suggests that ctDNA genotyping allows
non-invasive monitoring of response in lung cancer patients
receiving therapy (FIGS. 4A-4B). In FIG. 4A the patient received
treatment, but continued to progress, whereas patient in FIG. 4B
received treatment and responded.
[0100] Patients with EGFR-mutant lung cancer starting treatment
with EGFR-targeted therapy underwent serial monitoring of EGFR exon
19 and EGFR T790M plasma genotype. Responding patients had
normalization of their plasma genotype. When resistance developed,
the original EGFR mutation again became detected (dashed line) as
well as a new T790M resistance mutation (solid line). Genotyping of
the patient's tumor at time of progression also demonstrated an
acquired T790M resistance mutation. Intriguingly, plasma T790M was
detected 8 weeks prior to clinical progression. These findings
suggest serial ctDNA genotyping could allow monitoring for response
as well as assessment for new mutations when resistance develops
(FIG. 5A).
[0101] The signal for acquired resistant (solid line in FIG. 5A)
can be used to guide treatment with second generation therapies
(demonstrated in FIG. 5B). In that case the resistance biomarker is
used to change treatment and after treatment it becomes a marker to
monitor whether the treatment works (similar to the dashed line in
FIG. 5A). FIG. 6 shows more combinations of biomarkers.
REFERENCES
[0102] Ciriello et al., Mutual exclusivity analysis identifies
oncogenic network modules. Genome Res. 2012. 22: 398-406 [0103] The
Cancer Genome Atlas Research Network. 2008. Comprehensive genomic
characterization defines human glioblastoma genes and core
pathways. Nature 455: 1061-1068. [0104] The Cancer Genome Atlas
Research Network. 2011. Integrated genomic analyses of ovarian
carcinoma. Nature 474: 609-615. [0105] Cui Q, A network of cancer
genes with co-occurring and anti-co-occurring mutations. PLoS One.
2010 Oct. 4; 5(10).
Example 2: Noninvasive Detection of Response and Resistance in
EGFR-Mutant Lung Cancer Using Quantitative Next-Generation
Genotyping of Cell-Free Plasma DNA
Materials and Methods
Patient Population
[0106] For the primary study population, patients with advanced
NSCLC undergoing routine tumor genotyping were selected. All
patients consented to an IRB-approved protocol allowing collection
and genomic analysis of blood specimens, limited to <50 mL of
blood over any 3 month period. Patients were eligible for cfDNA
analysis if they harbored a known EGFR or KRAS mutation in their
NSCLC. Tumor genotyping of EGFR and KRAS was performed in a
clinical, CLIA-approved laboratory. A second population of patients
with advanced melanoma and a known BRAF genotype was also studied
after consent to specimen collection on an IRB-approved
protocol.
Plasma Collection
[0107] For each eligible patient, plasma was collected during
routine care either prior to first-line therapy or at a subsequent
time when the cancer was progressing on therapy. Additional
follow-up specimens were collected if possible during routine care.
Each specimen was collected into one 10 mL EDTA-containing
vacutainer and was spun into plasma within 4 hours of collection.
Plasma cfDNA was extracted and frozen at -80 C until genotyping.
Total DNA concentration in extracted plasma was measured via a
modified quantitative PCR assay for human long interspersed element
1 (LINE-1).
Droplet Digital PCR
[0108] Droplet Digital PCR (ddPCR) is a digital PCR technology that
takes advantage of developments in microfluids and surfactant
chemistries. Whereas conventional digital PCR involves a cumbersome
process of diluting input DNA into individual wells for analysis,
ddPCR emulsifies input DNA into .about.20,000 droplets that are PCR
amplified and fluorescently labeled, and then read in an automated
droplet flow cytometer (FIG. 1). Each droplet is individually
assigned a positive or negative value based on the fluorescent
intensity. The amount of positives and negatives are read by a flow
cytometer and are used to calculate the concentration of an allele.
To minimize bias and to ensure the integrity of results, the
laboratory was blinded to the tumor genotype when testing plasma
specimens, but results were selectively unblinded for data
analysis. Each plasma sample was analyzed in triplicate with an
increasing quantity of input DNA (e.g. 1 .mu.L, 2 .mu.L, and 4
.mu.L). Results were normalized to a mean concentration of mutant
alleles per L DNA input, and reported as copies of mutant allele
per 100 .mu.L of DNA, the approximate DNA quantity isolated from
one blood specimen.
Results
Assay Characteristics
[0109] Two assays for EGFR L858R and exon 19 deletions were first
developed; the latter assay was designed to detect loss of the
wildtype signal and therefore could detect deletions of variable
sequence. To demonstrate the analytical sensitivity and specificity
of each assay, each ddPCR cycling condition was optimized to yield
the maximum fluorescent signal with minimal increase in background
signal. For each TaqMan probe, the optimal annealing temperature
was determined by testing each assay across a temperature gradient
of 55.0 -65.degree. C. Using serial dilutions of mutant DNA, it was
found that ddPCR detects a mutation prevalence between 0.005% and
0.01% with a sensitivity of 5 to 50 mutant copies out of 10,000
(FIGS. 13A-13D), depending on the mutation assayed. Experiments
were repeated over three non-consecutive days. Both assays
demonstrated linear quantification of allelic prevalence across a
dynamic range spanning 4 orders of magnitude. From a technical
standpoint, this suggested that ddPCR provides a reliable and
quantitative measure of low prevalence EGFR mutant alleles within a
plasma sample.
Maximizing Positive Predictive Value
[0110] To optimize the specificity of the EGFR genotyping assays
(and utility in guiding clinical decisions), the incidence of false
positive reads was tested in a gold-standard negative population.
To ensure selection of patients certain to be wildtype for EGFR,
patients with KRAS-mutant lung cancers were studied. Large studies
have found that EGFR and KRAS mutations are non-overlapping in
NSCLC and represent distinct cancer populations, therefore any
EGFR-mutant DNA found in the plasma of patients with KRAS-mutant
NSCLC can be interpreted as biologically insignificant and
representative of the "normal range" for the assay.
[0111] The EGFR L858R assay was first studied in 23 NSCLC patients,
12 with EGFR L858R and 11 with KRAS mutations in their cancers. Low
levels of EGFR L858R were detected in 2 KRAS-mutant cases (18%)
with a peak level of 1.7 mutations/100 .mu.L of DNA (FIG. 9A).
Using 2 mutations/100 .mu.L of DNA as the threshold for a positive
result, 8 of 12 cases were correctly identified as positive for
EGFR L858R (66% sensitivity; 100% specificity). The variable exon
19 deletion assay was next studied in 23 NSCLC patients, 9 with
EGFR exon 19 deletions and 14 with KRAS mutations in their cancers.
Low levels of EGFR exon 19 deletions were detected in 3 KRAS-mutant
cases (21%) with a peak value of 9.9 mutations/100 .mu.L DNA (FIG.
9B). Using 12 mutations/100 .mu.L of DNA as the threshold for a
positive result, 6 of 9 cases were correctly identified as positive
for EGFR exon 19 deletion (66% sensitivity; 100% specificity).
Lastly, the reverse experiment was tested using a KRAS G12C assay
that was developed as above. Of 17 patients with EGFR-mutant lung
cancer, none had measurable mutant KRAS (FIG. 9C). Using a
threshold of 1 mutation/100 .mu.L of DNA, 11 of 14 KRAS G12C cases
were correctly identified as positive (79% sensitivity; 100%
specificity).
[0112] To gauge the generalizability of this assay to other
genotype-defined malignancies, an assay was developed for BRAF
V600E in the fashion described above and tested plasma specimens
from 13 melanoma patients. Using a threshold of 1 mutation/100
.mu.L of DNA for a positive result, we had a sensitivity of 86% and
specificity of 100% (FIG. 14), demonstrating potential value of
ddPCR genotyping in a disease other than NSCLC.
Quality Control to Improve Sensitivity
[0113] To better understand the false negative results in a subset
of cases, LINE-1 was measured to assess the quantity and quality of
cfDNA in each plasma specimen. LINE-1 is an easily measured,
genomically common retrotransposon that has been previously used to
estimate total DNA in plasma. The amplicons used for the LINE-1
qPCR assays are 82 bp and 107 bp, providing a snapshot of the
minimum size of DNA fragments. LINE-1 levels were first measured in
69 specimens and compared them to overall DNA concentration as
measured with PicoGreen (FIG. 10A) and found a high degree of
correlation (R.sup.2=0.94, p<0.0001). Median LINE-1
concentration was of 7700 pg/.mu.L (interquartile range: 3072-14415
pg/.mu.L) across 69 specimens.
[0114] LINE-1 levels were next measured in plasma specimens from 38
EGFR-mutant and KRAS-mutant lung cancer patients studied in the
above experiments. Detection of mutant alleles overall improved
with increased levels of LINE-1(FIG. 10B). In specimens with LINE-1
levels less than 3000 pg/.mu.L, representing a low concentration of
cfDNA, 50% had no detectable plasma genotype. Also observed was no
detection of plasma genotype in cases with the highest levels of
LINE-1 (greater than 700,000 pg/.mu.L), likely indicating a high
level of germline DNA obscuring detection of mutant cfDNA. However,
when considering only cases with a LINE-1 concentration between
3000 and 700,000 pg/.mu.L, sensitivity was 100% with 100%
specificity (FIG. 10B), indicating that LINE-1 can be used for
quality control to clarify which specimens are less likely to have
a falsely negative result.
Developing a Disease Monitoring Biomarker
[0115] To assess the value of cfDNA genotype prevalence as a
disease monitoring biomarker, the range of variability was
quantified. Using the techniques described above, a fifth
genotyping assay was developed to detect the EGFR T790M mutation.
Human plasma DNA specimens were generated that contained either 1,
2, 10, or 20 copies of EGFR T790M per 25 .mu.L reaction, divided
each into 32 individual specimens, and each of these were tested
for T790M prevalence by ddPCR. The assay exhibited a Poisson
distribution between positives droplets and sample input with
acceptable coefficient of variance in the range of 20-30% (FIGS.
15A-15B), suggesting that changes exceeding this amount represent a
true change in tumor burden or biology.
[0116] To gauge feasibility, serial plasma specimens were studied
from patients with genotype-defined lung cancer or melanoma to
determine whether changes in cfDNA were representative of tumor
biology (FIGS. 11A-11D). In a patient with EGFR-mutant NSCLC
receiving chemotherapy after failing erlotinib (FIG. 11A), an
increase in plasma L858R and T790M was seen with development of new
brain metastases, followed by decreased plasma levels when
treatment on a clinical trial was initiated. In a second case of
EGFR-mutant NSCLC receiving chemotherapy (FIG. 11B), plasma L858R
decreased as the patient's pleural drainage resolved, though CT
imaging of the non-measurable disease showed disease stability. In
a patient with KRAS-mutant NSCLC and bone metastases (FIG. 11C),
chemotherapy caused a decrease in plasma G12C levels concordant
with improved pain control and decreased opiate requirement.
Lastly, a patient with BRAF-mutant melanoma had progression on
experimental immune therapy followed by response to vemurafenib
(FIG. 11D), seen in the rise and fall of plasma V600E levels. These
experiments demonstrated that cfDNA genotyping has value for serial
assessment of disease status, even in patients without objectively
measurable disease on CT.
Monitoring for Resistance Mutations
[0117] To determine whether ddPCR could identify the development of
resistance mutations after treatment with targeted therapy,
patients were studied with advanced EGFR-mutant NSCLC treated on a
prospective clinical trial of first-line erlotinib (NCT00997334),
limiting the analysis to 13 patients that had serial plasma
specimens collected until development of objective progression per
the Response Evaluation Criteria In Solid Tumors (RECIST). In each
of these patients, genotyping of archived tissue at diagnosis
identified an EGFR exon 19 deletion without evidence of T790M. Four
patients had no detectable pretreatment plasma genotype and were
excluded, leaving 9 cases (69%) for analysis.
[0118] All 9 patients exhibited a plasma response to erlotinib,
with 8 demonstrating a complete plasma response (FIGS. 12A-12F). In
6 of the patients, plasma levels of mutant EGFR were again detected
at objective progression, with plasma progression detected 4-12
weeks prior to RECIST progression. In each of these patients,
plasma T790M could also be identified at progression, generally at
somewhat lower levels than the EGFR sensitizing mutation. Four of
these patients had a tumor rebiopsy adequate for EGFR genotyping,
and T790M was confirmed in each. The remaining three patients had
no reemergence of plasma genotype at objective progression;
notably, each of these patients had indolent asymptomatic
progression in the chest only, such that they subsequently
continued single-agent erlotinib off-protocol.
Discussion
[0119] Described herein is a new quantitative assay for
plasma-based tumor genotyping which has been technically optimized
for translation into clinical practice. By quantifying the
prevalence of targetable genotypes in clinical plasma specimens,
and through study of rigorous gold-standard negative cases
harboring non-overlapping cancer genotypes, a normal range has been
identified for EGFR and KRAS mutation detection using ddPCR. Using
such a calculated threshold as the criteria for a positive results,
as well as LINE-1 concentration to eliminate poor quality
specimens, the data demonstrates that this assay has high
sensitivity and specificity.
[0120] Because many targetable genotypes are relatively uncommon,
assay development was focused on maximizing specificity. Consider,
for example, a plasma assay for detecting EGFR sensitizing
mutations, present in 8.6% of 10,000 NSCLC patients from the large
French experience (Barlesi F, Blons H, Beau-Faller M, Rouquette I,
Ouafik Lh, Mosser J, et al. Biomarkers (BM) France: Results of
routine EGFR, HER2, KRAS, BRAF, PI3KCA mutations detection and
EML4-ALK gene fusion assessment on the first 10,000 non-small cell
lung cancer (NSCLC) patients (pts). ASCO Meeting Abstracts. 2013;
31:8000). In this population, a plasma assay for EGFR mutations
having 80% sensitivity and 90% or 95% specificity would have a PPV
of only 43% or 60%, respectively. For this reason, a clinical-grade
assay will likely need to sacrifice sensitivity in order to
optimize specificity. In the same population, an assay with 70%
sensitivity and 99% or 100% specificity would result in a PPV of
87% or 100%, respectively. Furthermore, the need to maximize
specificity is magnified when testing for rarer genotypes such as
BRAF V600E in NSCLC, representing only 2% of patients. One valuable
characteristic of a quantitative assay such as ddPCR is the
flexibility to allow an alteration of the criterion for positive if
the pretest probability changes (e.g. Asian lung cancer patients).
This is in contrast to an allele-specific PCR assay, such as one
which showed high concordance with tumor genotyping in a
preliminary analysis of plasma from 241 Asian lung cancer patients
(Mok T, Wu Y L, Lee J S, Yu C-J, Sriuranpong V, Wen W, et al.
Detection of EGFR-activating mutations from plasma DNA as a potent
predictor of survival outcomes in FASTACT 2: A randomized phase III
study on intercalated combination of erlotinib (E) and chemotherapy
(C). ASCO Meeting Abstracts. 2013; 31:8021); as such an assay is
qualitative, it cannot easily be adjusted to a higher specificity
criterion in populations with lower mutation prevalence.
[0121] This study allows identification of the acquisition of
plasma T790M in lung cancer patients prior to clinical development
of resistance to EGFR kinase inhibitors. This has particular
importance given the growing role of EGFR T790M as a biomarker for
patients with EGFR-mutant lung cancer and acquired resistance.
Firstly, acquired T790M has been associated with indolent growth
and a favorable prognosis compared to T790M-negative acquired
resistance. Secondly, third-generation EGFR kinase inhibitors with
T790M-specific activity have recently been shown to induce
responses in some patients. While pharmaceutical development of
T790M-directed targeted therapies could be limited by the
challenges of performing a repeat biopsy after resistance develops,
the data described herein indicates that emergence of EGFR T790M
can be identified noninvasively using ddPCR, and potentially used
to guide subsequent treatment.
[0122] The quantitative nature of plasma genotyping with ddPCR also
offers a mechanism for monitoring the prevalence of tumor clones
harboring a specific genotype, potentially giving insight into the
pharmacodynamics of a targeted therapy. In liquid malignancies like
chronic myelogenous leukemia, rapidity of molecular response to
kinase inhibitors has been established as an important biomarker of
prognosis, and helps indicate which patients may need early salvage
therapy. Similarly, plasma response to targeted therapies may prove
to be valuable biomarker for genotype-defined solid tumors, both as
a clinical biomarker of favorable outcome and potentially as an
early clinical trial endpoint. Indeed, this was demonstrated in the
small series described herein--the one patient not exhibiting a
complete plasma response to erlotinib had early progression. In
addition, response assessment using plasma genotype quantification
could potentially allow trial accrual for those patients with
genotype-defined solid tumors that are not objectively measurable
using conventional response criteria.
Methods
[0123] Patients were identified from four IRB-approved protocols on
the basis of (I) advanced NSCLC, (II) acquired resistance to an
EGFR TKI, (III) possessing or having a planned re-biopsy and (IV)
consent to research blood draws.
[0124] Baseline blood samples were collected from each patient in a
standard EDTA tube. A subset of patients initiating a new treatment
at the time of initial draw underwent two subsequent blood draws
after the first and second cycles of treatment. Plasma was prepared
using a modified protocol to minimize cell rupture. cfDNA was
extracted using the QIAmp circulating nucleic acid kit (Qiagen).
Previously validated probes and ddPCR system (BioRads) were used to
detect and quantify EGFR mutation concentration as previously
described (Oxnard et al., Clinical Cancer Research, 2014). The
threshold for a positive test result was specific to each EGFR
mutation studied: exon 19 del=6 copies/mL, L858R=1 copy/mL,
T790M=0.5 copies/mL. Patient characteristics are shown in Table
2.
TABLE-US-00006 TABLE 2 Patient characteristics N = 45 Median age 57
(26-80) Sex Female 36 (80%) Male 9 (20%) Stage IV 41 (91%)
Recurrent 4 (9%) Distant metastases Brain 7 (15%) Bone 21 (47%)
Visceral 10 (22%) Sensitizing mutation Exon 19 del 33 (73%) L858R 9
(20%) Other 3 (7%) Treatment (N = 12).sup..dagger. Chemotherapy 2
Immunotherapy 1 Investigational drug therapy 9 .sup..dagger.For
patients starting a new therapy with serial plasma collected.
TABLE-US-00007 TABLE 3 PPV.sup.1 Specificity.sup.2
Sensitivity.sup.3 EGFR T790M All stage IV 95% (19/20) .sup.4 93%
(13/14) .sup.4 73% (19/26) Stage IVa 100% (2/2) 100% (8/8) 67%
(2/3) Stage IVb 94% (17/18) 84% (5/6) 73% (17/23) EGFR sensitizing
mutation.sup.5 All stage IV -- -- 58% (25/43) Stage IVa -- -- 36%
(5/14) Stage IVb -- -- 69% (20/29) .sup.1Positive predictive value
(PPV) = true positive/(true positive + false positive)
.sup.2Specificity = true negative/(true negative + false positive)
.sup.3Sensitivity = true positive/(true positive + false negative)
.sup.4 Single false positive case had 4 copies/mL of T790M and 208
copies/mL of exon 19 del, with exon 19 del only on pleural biopsy
.sup.5EGFR exon 19 del & L858R, as all patients were mutation
positive, specificity and positive predictive value cannot be
calculated
Results
[0125] In patients with at least a minor response to treatment
(defined as >10% reduction in tumor mass on initial re-staging
CT scan), plasma genotype concentration (includes both EGFR exon 19
del and L858R depending on individual patient genotype) decreases
an average of 1773 fold (FIG. 16B). Plasma genotype concentration
is stable or increases in patients without evidence of a response
(FIG. 16A). The sensitivity of ddPCR-based plasma genotyping may be
better in patients with extra-thoracic metastases (stage IVb).
[0126] Case Report: Plasma Genotyping Directed Treatment
[0127] Plasma genotyping in a patient with acquired resistance to
EGFR TKI detects EGFR T790M 24 days earlier than re-biopsy and
tissue genotyping. On Day 0, when CT shows marked progression on
erlotinib, plasma is drawn (FIG. 17). On DAY 1, cfDNA genotyping
detects 806 copies/ml of EGFR T790M. On DAY 25, report from
rebiopsy genotyping shows EGFR T790M. Thus, this technology has the
potential to allow treatment to begin weeks earlier without the
risks of a biopsy. On DAY 31, Patient starts treatment with an
investigational drug therapy. On DAY 73, CT demonstrates a
radiographic response
[0128] In conclusion, described herein is a cfDNA genotyping assay
that is optimized for clinical application. Droplet Digital PCR has
a rapid turnaround time, can be performed on routine plasma
specimens, is relatively inexpensive, and provides results with a
wide dynamic range, making it a an attractive tool for both
clinical care and for clinical research.
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[0154] Various modifications of the invention in addition to those
shown and described herein will become apparent to those skilled in
the art from the foregoing description and fall within the scope of
the appended claims. The advantages and objects of the invention
are not necessarily encompassed by each embodiment of the
invention.
Sequence CWU 1
1
4123DNAArtificial SequenceSynthetic Oligonucleotide 1gtgagaaagt
taaaattccc gtc 23219DNAArtificial SequenceSynthetic Oligonucleotide
2cacacagcaa agcagaaac 19322DNAArtificial SequenceSynthetic
Oligonucleotide 3aggaattaag agaagcaaca tc 22419DNAArtificial
SequenceSynthetic Oligonucleotide 4atcgaggatt tccttgttg 19
* * * * *