U.S. patent application number 13/579964 was filed with the patent office on 2013-08-15 for personalized tumor biomarkers.
This patent application is currently assigned to THE JOHNS HOPKINS UNIVERSITY. The applicant listed for this patent is Luis Diaz, Kenneth W. Kinzler, Rebecca J. Leary, Victor Velculescu, Bert Volgelstein. Invention is credited to Luis Diaz, Kenneth W. Kinzler, Rebecca J. Leary, Victor Velculescu, Bert Volgelstein.
Application Number | 20130210645 13/579964 |
Document ID | / |
Family ID | 44483555 |
Filed Date | 2013-08-15 |
United States Patent
Application |
20130210645 |
Kind Code |
A1 |
Volgelstein; Bert ; et
al. |
August 15, 2013 |
PERSONALIZED TUMOR BIOMARKERS
Abstract
Clinical management of human cancer is dependent on the accurate
monitoring of residual and recurrent tumors. We have developed a
method, called personalized analysis of rearranged ends (PARE),
which can identify translocations in solid tumors. Analysis of four
colorectal and two breast cancers revealed an average of nine
rearranged sequences (range 4 to 15) per tumor. Polymerase chain
reaction with primers spanning the breakpoints were able to detect
mutant DNA molecules present at levels lower than 0.001% and
readily identified mutated circulating DNA in patient plasma
samples. This approach provides an exquisitely sensitive and
broadly applicable approach for the development of personalized
biomarkers to enhance the clinical management of cancer
patients.
Inventors: |
Volgelstein; Bert;
(Baltimore, MD) ; Kinzler; Kenneth W.; (Baltimore,
MD) ; Velculescu; Victor; (Dayton, MD) ; Diaz;
Luis; (Ellicott City, MD) ; Leary; Rebecca J.;
(Baltimore, MD) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Volgelstein; Bert
Kinzler; Kenneth W.
Velculescu; Victor
Diaz; Luis
Leary; Rebecca J. |
Baltimore
Baltimore
Dayton
Ellicott City
Baltimore |
MD
MD
MD
MD
MD |
US
US
US
US
US |
|
|
Assignee: |
THE JOHNS HOPKINS
UNIVERSITY
Baltimore
MD
|
Family ID: |
44483555 |
Appl. No.: |
13/579964 |
Filed: |
February 17, 2011 |
PCT Filed: |
February 17, 2011 |
PCT NO: |
PCT/US11/25152 |
371 Date: |
January 7, 2013 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
61305589 |
Feb 18, 2010 |
|
|
|
Current U.S.
Class: |
506/4 ; 435/6.11;
536/24.33 |
Current CPC
Class: |
C12Q 1/6886 20130101;
C12Q 2600/156 20130101 |
Class at
Publication: |
506/4 ; 435/6.11;
536/24.33 |
International
Class: |
C12Q 1/68 20060101
C12Q001/68 |
Goverment Interests
[0001] This invention was made with government support under grants
CA121113, CA057345, CA62924, and CA043460 awarded by the National
Institutes of Health. The government has certain rights in the
invention.
Claims
1. A method of identifying a personalized tumor marker for a cancer
patient, comprising: making a mate-paired library from tumor DNA of
the patient, wherein mate pairs of said library comprise two
genomic tags that are co-linear but not contiguous in a segment of
the tumor DNA; determining sequence of a plurality of mate pairs of
the library; determining regions of copy number differences among
regions in the tumor DNA of the patient; identifying mate paired
tags which map within a region of copy number difference or
spanning a boundary of copy number difference as potential markers
of a tumor-specific DNA rearrangement in the cancer patient.
2. The method of claim 1 further comprising: identifying a
breakpoint between, within, or spanning the regions of copy number
differences and designing amplification primers that hybridize to
priming sites that flank the breakpoint, wherein the priming sites
are separated by between 20 and 200 basepairs in the tumor DNA.
3. The method of claim 1 further comprising: testing identified
paired tags by comparing to non-tumor DNA or to sequence of
non-tumor DNA, and confirming a tumor-specific DNA rearrangement if
the two genomic tags of a mate paired tag are at different
locations or in a different orientation within a chromosome or on
different chromosomes of non-tumor DNA compared to tumor DNA.
4. The method of claim 3 wherein the non-tumor DNA is from the
cancer patient.
5. The method of claim 3 further comprising: identifying a
breakpoint in the DNA rearrangement and designing amplification
primers that hybridize to priming sites that flank the breakpoint,
wherein the priming sites are separated by less than 200 basepairs
in the tumor DNA.
6. The method of claim 2 or 5, further comprising: amplifying a DNA
fragment using a template from the patient's tissues or body fluids
and said amplification primers; determining the amount or
proportion of amplified DNA fragment in the patient's tissue or
body fluid, wherein said amount is an indication of tumor
burden.
7. A method of assessing or detecting tumor in a patient,
comprising: amplifying a DNA fragment using a template from the
patient's tissues or body fluids and primers that span a
patient-specific, tumor-specific rearrangement breakpoint, wherein
the rearrangement breakpoint is between genes or gene loci involved
in rearrangements in <1% of tumors of patients with the same
type of tumor; determining the amount or proportion of amplified
DNA fragment in the patient's tissue or body fluid.
8. The method of claim 7 wherein the rearrangement breakpoint is
between genes or gene loci involved in rearrangements in <0.1%
of tumors of patients with the same type of tumor.
9. The method of claim 7 wherein the rearrangement breakpoint is
not associated with a leukemia, lymphoma, sarcoma, or prostate
cancer.
10. The method of claim 7 wherein the step of determining is
performed with samples obtained from the cancer patient at a
plurality of times.
11. The method of claim 10 wherein the plurality of times are
before and during an anti-tumor therapy.
12. The method of claim 10 wherein the plurality of times are
during an anti-tumor therapy.
13. The method of claim 10 wherein the plurality of times are
before and after surgery.
14. The method of claim 10 wherein the plurality of times are to
monitor a patient in remission for relapse or recurrence.
15. The method of claim 7 wherein presence of an amount of
amplified DNA fragment indicates residual disease.
16. The method of claim 7 wherein presence of an amount of
amplified DNA fragment in a tumor margin indicates incomplete
surgical resection.
17. The method of claim 7 wherein the template is from the
patient's lymph nodes.
18. The method of claim 7 wherein the template is from the
patient's blood.
19. A method of identifying a personalized tumor marker for a
cancer patient, comprising: determining sequence of two ends of
each of a plurality of fragments of DNA from the cancer patient;
determining regions of copy number differences among regions in the
tumor DNA of the patient; identifying fragments of the plurality of
fragments which map within a region of copy number difference or
spanning a boundary of copy number difference as potential markers
of a tumor-specific DNA rearrangement in the cancer patient.
20. The method of claim 19 further comprising: identifying a
breakpoint between, within, or spanning the regions of copy number
differences and designing amplification primers that hybridize to
priming sites that flank the breakpoint, wherein the priming sites
are separated by between 20 and 200 basepairs in the tumor DNA.
21. The method of claim 19 further comprising: testing two ends of
a fragment by comparing to non-tumor DNA or to sequence of
non-tumor DNA; and identifying a tumor-specific DNA rearrangement
if the two ends are at different locations or in a different
orientation within a chromosome or on different chromosomes of
non-tumor DNA compared to tumor DNA.
22. The method of claim 21 wherein the non-tumor DNA is from the
cancer patient.
23. The method of claim 21 further comprising: identifying a
breakpoint in the DNA rearrangement and designing amplification
primers that hybridize to priming sites that flank the breakpoint,
wherein the priming sites are separated by less than 200 basepairs
in the tumor DNA.
24. The method of claim 20 or 23 further comprising: amplifying a
DNA fragment using a template from the patient's tissues or body
fluids and said amplification primers; determining the amount or
proportion of amplified DNA fragment in the patient's tissue or
body fluid, wherein said amount is an indication of tumor
burden.
25. A method of identifying a personalized tumor marker for a
cancer patient, comprising: testing a plurality of mate paired tags
of a library of mate paired tags by comparing to non-tumor DNA or
to sequence of non-tumor DNA, wherein each of said mate paired tags
comprises two genomic tags that are co-linear but not contiguous in
a segment of tumor DNA of the cancer patient; and identifying a
tumor-specific DNA rearrangement if the two genomic tags of a mate
paired tag are at different locations or in a different orientation
within a chromosome or on different chromosomes of non-tumor DNA
compared to tumor DNA.
26. The method of claim 25 further comprising: identifying a
breakpoint in the DNA rearrangement and designing amplification
primers that hybridize to priming sites that flank the breakpoint,
wherein the priming sites are separated by less than 200 basepairs
in the tumor DNA.
27. The method of claim 25 wherein prior to the step of testing the
mate paired tags are identified as being within regions of copy
number difference relative to adjacent regions of the genome.
28. The method of claim 25 wherein prior to the step of testing the
mate paired tags are enriched for tags from regions of copy number
difference relative to adjacent regions of the genome.
29. A method of identifying a personalized tumor marker for a
cancer patient, comprising: testing two ends of a plurality of
fragments of tumor DNA of the cancer patient by comparing to
non-tumor DNA or to sequence of non-tumor DNA; and identifying a
tumor-specific DNA rearrangement if the ends of a fragment are at
different locations or in a different orientation within a
chromosome or on different chromosomes of non-tumor DNA compared to
tumor DNA.
30. The method of claim 29 further comprising: identifying a
breakpoint in the DNA rearrangement and designing amplification
primers that hybridize to priming sites that flank the breakpoint,
wherein the priming sites are separated by less than 200 basepairs
in the tumor DNA.
31. The method of claim 29 wherein prior to the step of testing the
plurality of fragments are identified as being within regions of
copy number difference relative to adjacent regions of the
genome.
32. The method of claim 29 wherein prior to the step of testing the
plurality of fragments are enriched for tags from regions of copy
number difference relative to adjacent regions of the genome.
33. A method of screening for a cancer in a human, comprising:
testing a plurality of mate paired tags of a library of mate paired
tags by comparing to normal DNA or to sequence of normal DNA,
wherein each of said mate paired tags comprises two genomic tags
that are co-linear but not contiguous in a segment of DNA in the
blood of the human; and identifying DNA rearrangement if the two
genomic tags of a mate paired tag are at different locations or in
a different orientation within a chromosome or on different
chromosomes of normal DNA compared to blood DNA, wherein the
presence of a DNA rearrangement suggests the presence of a cancer
in the human.
34. A method of screening for a cancer in a human, comprising:
testing two ends of a fragment of blood DNA of the human by
comparing to normal DNA or to sequence of normal DNA; and
identifying a DNA rearrangement if the ends are at different
locations or in a different orientation within a chromosome or on
different chromosomes of normal DNA compared to blood DNA, wherein
the presence of a DNA rearrangement suggests the presence of a
cancer in the human.
35. The method of claim 33 or 34 wherein the normal DNA is
lymphocyte DNA.
36. The method of claim 33 or 34 wherein the normal DNA is buccal
DNA.
37. The method of claim 25 or 29 wherein the patient has a cancer
that is not a leukemia, lymphoma, sarcoma, or prostate cancer.
38. A kit for monitoring presence or amount of a breakpoint in a
somatic DNA rearrangement in tumor DNA of a patient, comprising:
one or more pairs of amplification primers, wherein each pair is
complementary to priming sites on opposite sides of a breakpoint,
wherein the priming sites are separated by less than 200 basepairs
in the tumor DNA, and wherein the DNA rearrangement occurs in
<1% of tumors of patients with the same type of tumor.
39. The kit of claim 38 wherein the tumor DNA is not from a
leukemia, lymphoma, sarcoma, or prostate cancer.
40. The kit of claim 38 wherein the DNA rearrangement occurs in
<0.1% of tumors of patients with the same type of tumor.
41. The kit of claim 38 wherein the kit comprises a plurality of
pairs of amplification primers and the plurality of pairs are
complementary to priming sites on opposite sides of a plurality of
breakpoints in tumor DNA of the patient.
42. The kit of claim 38 wherein the kit further comprises a DNA
polymerase for amplification.
43. The kit of claim 38 wherein the kit further comprises
deoxyribonucleotides for amplification substrates.
44. The kit of claim 38 wherein the kit further comprises reagents
for preparing template from tumor DNA of the patient.
Description
TECHNICAL FIELD OF THE INVENTION
[0002] This invention is related to the area of cancer detection
and management. In particular, it relates to identification and use
of somatic rearrangements as markers of a person's cancer.
BACKGROUND OF THE INVENTION
[0003] A nearly universal feature of human cancer is the widespread
rearrangement of chromosomes as a result of chromosomal instability
(1). Such structural alterations begin to occur at the earliest
stages of tumorigenesis and persist throughout tumor development.
The consequences of chromosomal instability can include copy number
alterations (duplications, amplifications and deletions),
inversions, insertions, and translocations (2). Historically, the
ability to detect such alterations has been limited by the
resolution of genetic analyses. However, a number of more recent
approaches including high density oligonucleotide arrays and high
throughput sequencing have allowed detection of changes at much
higher resolution (3-15).
[0004] Tumor-specific (somatic) chromosomal rearrangements have the
potential to serve as highly sensitive biomarkers for tumor
detection. Such alterations are not present in normal cells and
should be exquisitely specific. Rearrangement-associated biomarkers
therefore offer a reliable measure that would be useful for
monitoring tumor response to specific therapies, detecting residual
disease after surgery, and for long-term clinical management.
Recurrent somatic structural alterations, such as those involving
the BCR-ABL oncogene (the target of the Philadelphia chromosome
translocation), immunoglobulin (Ig) genes, T cell receptor (TCR)
genes, and the retinoic acid receptor alpha (RAR.alpha.) gene, have
been shown to be useful as diagnostic markers in certain
hematopoietic malignancies (16-20). However, recurrent structural
alterations do not generally occur in most solid tumors. There is a
continuing need in the art to develop tools for diagnosing and
monitoring cancers.
SUMMARY OF THE INVENTION
[0005] According to one aspect of the invention a method is
provided for identifying a personalized tumor marker for a cancer
patient. A mate-paired library is made from tumor DNA of the
patient. Mate pairs of the library comprise two genomic tags that
are co-linear but not contiguous in a segment of the tumor DNA.
Sequence of a plurality of mate pairs of the library is determined.
Regions of copy number differences among regions in the tumor DNA
of the patient are determined. Mate paired tags which map within a
region of copy number difference or spanning a boundary of copy
number difference are identified as potential markers of a
tumor-specific DNA rearrangement in the cancer patient.
[0006] According to another aspect of the invention a method is
provided for assessing or detecting tumor in a patient. A DNA
fragment is amplified using a template from the patient's tissues
or body fluids and primers that span a patient-specific,
tumor-specific rearrangement breakpoint. The rearrangement
breakpoint is between genes involved in rearrangements in <1% of
tumors of patients with the same type of tumor. The amount or
proportion of amplified DNA fragment in the patient's tissue or
body fluid is determined.
[0007] Another aspect of the invention is another method of
identifying a personalized tumor marker for a cancer patient.
Sequence of two ends of each of a plurality of fragments of DNA
from the cancer patient is determined. Regions of copy number
differences among regions in the tumor DNA of the patient are
determined. Fragments of the plurality of fragments which map
within a region of copy number difference or spanning a boundary of
copy number difference are identified as potential markers of a
tumor-specific DNA rearrangement in the cancer patient.
[0008] A further aspect of the invention is another method of
identifying a personalized tumor marker for a cancer patient. A
plurality of mate paired tags of a library of mate paired tags is
tested by comparing to non-tumor DNA or to sequence of non-tumor
DNA. Each of the mate paired tags comprises two genomic tags that
are co-linear but not contiguous in a segment of tumor DNA of the
cancer patient. A tumor-specific DNA rearrangement is identified if
the two genomic tags of a mate paired tag are at different
locations or in a different orientation within a chromosome or on
different chromosomes of non-tumor DNA compared to tumor DNA.
[0009] Yet another aspect of the invention is another method of
identifying a personalized tumor marker for a cancer patient. Two
ends of a plurality of fragments of tumor DNA of the cancer patient
are tested by comparing to non-tumor DNA or to sequence of
non-tumor DNA. A tumor-specific DNA rearrangement is identified if
the ends of a fragment are at different locations or in a different
orientation within a chromosome or on different chromosomes of
non-tumor DNA compared to tumor DNA.
[0010] Still another aspect of the invention is a method of
screening for a cancer in a human. A plurality of mate paired tags
of a library of mate paired tags is tested by comparing to normal
DNA or to sequence of normal DNA. Each of the mate paired tags
comprises two genomic tags that are co-linear but not contiguous in
a segment of DNA in the blood of the human. A DNA rearrangement is
identified if the two genomic tags of a mate paired tag are at
different locations or in a different orientation within a
chromosome or on different chromosomes of normal DNA compared to
blood DNA. The presence of a DNA rearrangement suggests the
presence of a cancer in the human.
[0011] A further aspect of the invention is a method of screening
for a cancer in a human. Two ends of a fragment of blood DNA of the
human are tested by comparing to normal DNA or to sequence of
normal DNA. A DNA rearrangement is identified if the ends are at
different locations or in a different orientation within a
chromosome or on different chromosomes of normal DNA compared to
blood DNA. The presence of a DNA rearrangement suggests the
presence of a cancer in the human.
[0012] An additional aspect of the invention is a kit for
monitoring presence or amount of a breakpoint in a somatic DNA
rearrangement in tumor DNA of a patient. The kit may comprise one
or more pairs of amplification primers. Each pair is complementary
to priming sites on opposite sides of a breakpoint. The priming
sites are separated by less than 200 basepairs in the tumor DNA.
The DNA rearrangement occurs in <1% of tumors of patients with
the same type of tumor.
[0013] These and other embodiments which will be apparent to those
of skill in the art upon reading the specification provide the art
with methods for detecting and monitoring cancers in the body.
BRIEF DESCRIPTION OF THE DRAWINGS
[0014] FIG. 1. Schematic of "Personalized Analysis of Rearranged
Ends (PARE)" approach. The method is based on next generation
mate-paired analysis of e.g., resected tumor DNA to identify
individualized tumor-specific rearrangements. Such alterations are
used to develop PCR based quantitative analyses for personalized
tumor monitoring of plasma samples or other bodily fluids.
[0015] FIGS. 2A and 2B. Detection of tumor-specific rearrangements
in breast and colorectal cancers. Two representative rearrangements
are shown for each tumor sample, PCR amplification across
breakpoint regions is indicated in (FIG. 2A) and the genomic
coordinates for a representative mate-pair of each rearrangement
are listed in (FIG. 2B).
[0016] FIG. 3. Detection of tumor specific rearrangements in
mixtures of tumor and normal DNA. Decreasing amounts of tumor DNA
were mixed with increasing amounts of normal tissue DNA (300 ng
total) and were used as template molecules for PCR using
chromosomes 4/8 translocation specific primers (top) or chromosome
3 control primers (see Example 1 for additional information).
[0017] FIG. 4A-4B. Detection of tumor-specific rearrangements in
plasma of cancer patients. FIG. 4A. The identified chromosome 4/8
and 16 rearrangements were used to design PCR primers spanning
breakpoints and used to amplify rearranged DNA from tumor tissue
and plasma from patients Hx402 and Hx403, respectively. A plasma
sample from an unrelated healthy individual was used a control for
both rearrangements. FIG. 4B. Plasma samples from patient Hx402
were analyzed at different time points using digital PCR to
determine the fraction of genomic equivalents of plasma DNA
containing the chromosome 4/8 rearrangement. The fraction of
rearranged DNA at day 137 was 0.3%, consistent with residual
metastatic lesions present in the remaining lobe of the liver.
[0018] FIG. 5 (Figure S1.) Flow chart of approach used to identify
rearranged sequences
[0019] FIG. 6 (Figure S2.) Comparison of Digital Karyotyping,
Illumina SNP array, and SOLiD sequencing results on chromosome
8.
DETAILED DESCRIPTION OF THE INVENTION
[0020] We have found that any structural alteration identified in
an individual's tumor can be used as a tumor marker, even if it is
not found in tumors of the same type in other individuals and even
if it is not a "driver"--causing a selective growth advantage--but
merely a "passenger." Moreover, such markers can be used to detect
tumor and or quantify the tumor burden in an individual by
assessment of blood.
[0021] Somatic rearrangements are a focus of the present invention.
Such rearrangements are used as markers of a tumor. In particular,
the boundaries of the rearrangements can be detected and used as a
quantitative or qualitative indicator of the tumor. Because the
boundaries are unique to the tumor DNA, they should be exquisitely
specific markers of the tumor. Somatic rearrangements can be
detected using any method known in the art. One particularly useful
method is a technique called digital karyotyping. This technique
identifies changes in copy number across regions or windows in the
genome. Other methods may employ commercially available arrays to
detect regions of copy number differences among regions of a
genome. The copy number differences reflect a rearrangement, such
as a deletion or amplification, and an amplification can further
harbor other rearrangements within it. Once a somatic rearrangement
is identified, one or more of its boundaries (also referred to as
breakpoints) can be identified and that boundary can be a very
specific marker for the tumor. Identifying a boundary can be
accomplished by a number of techniques.
[0022] In one technique mate-paired genomic tags are tested to
determine different copy numbers of one member of the pair compared
to the other. A different copy number between two members suggests
that the tags span a rearrangement breakpoint or boundary. The
mate-pairs are typically derived from a single fragment that is
processed to yield two smaller portions that can be more readily
sequenced or analyzed. An intervening segment is typically removed,
leaving the two smaller portions linked on a single molecule in the
same orientation that they were found in the tumor genome.
[0023] A similar technique does not involve mate-pairs but involves
sequencing and/or analyzing two different portions or ends of a
single fragment of genomic DNA from a tumor. The two portions or
ends may be separated by any distance, from immediately adjacent up
to 1 kb, 1.5 kb, 2 kb, or 3 kb, for example. The ends may not be
the literal ends of a fragment, but may be close to the ends or
merely two non-overlapping portions. The sequence of the two ends
may be determined separately, for example from either end, or the
sequence can be determined in one direction and analyzed for
separate, non-overlapping segments of differing copy numbers.
[0024] Amplification primers are known in the art and typically
comprise between 15 and 50 nucleotides which are complementary to a
template. A pair of primers is complementary to opposite strands of
a template and can amplify a double stranded fragment that contains
the two primer sequences in addition to sequences which are between
them on the template. From 0 to 10, 20, 50, 100, 200, 500, 1000,
1500, or 2000 basepairs or nucleotides may lie between the two
primer-complementary sequences on the template. According to the
invention, each primer will hybridize to opposite sides of a
rearrangement boundary. These primers are also referred to as
spanning or flanking the breakpoint, because the amplicon that they
generate will span and/or flank the breakpoint. Optionally, a
primer may contain the boundary junction. Primers need not be 100%
complementary to template, but may incorporate other bases or
sequences of bases for other purposes, such as to facilitate
purification or downstream processing.
[0025] Once tumor-specific breakpoints are ascertained for an
individual patient, primers can be prepared and shipped elsewhere
for use. For example pairs or panels of pairs of primers can be
packaged in a single or divided container. The primers can be in
any suitable condition, including in solution, dried, freeze dried,
at room temperature, on wet ice, and on dry ice. Additional
components may be included in the kits, for example other reagents
for performing the monitoring or assessing with the primers.
Additional components may include a polymerase for amplification,
reagents for preparing template from cancer cells, normal cells, or
body fluids, control primers, control templates, labeled or
unlabelled deoxyribonucleotides.
[0026] In order to identify or confirm a rearrangement in tumor
DNA, tumor sequences can be compared to a reference sequence, for
example in a database, or to a sequence from normal DNA of the same
or a related individual. Two mate-paired tags or two fragment ends
that map to different locations on a chromosome or to different
chromosomes or to differently oriented sequences on the same
chromosome indicate a rearrangement. The comparison can be done in
silico or in vitro.
[0027] Breakpoints in a rearrangement are places where two
sequences are joined in a tumor DNA that are not joined in normal
or reference DNA. Thus the breakpoint refers to an inferred break
that occurred in order to join the sequences that are found in the
tumor DNA. Breakpoints are also referred to as boundaries of a
rearrangement. Normal DNA may be obtained from lymphocytes or a
buccal swab, for example. In cases where the subject has a
diagnosed tumor, normal DNA can be obtained from any non-tumor
tissue, including a matched tissue from the same organ.
[0028] The breakpoints which are of interest in the present methods
are those which are not known to be associated with or causative of
leukemia, lymphoma, sarcoma, or prostate cancers. The breakpoints
which are associated with or causative of those cancers typically
occur in a high proportion of such tumors, often between the same
or a limited number of genes or gene loci. The rearrangements used
in the present methods are more idiosyncratic, occurring between
the same genes or gene loci in less than 1%, less than 0.1%, or
less than 0.01% of the patients with the same type of tumor.
[0029] Assays using tumor-specific primers can be used for a
variety of purposes. For example, patients can be monitored over
time to see if a tumor is in remission or is progressing. The assay
can be used before, during, and/or after a therapy regimen. The
assay can be used to assess surgical efficacy. Tumor margins can be
assessed to guide the extent of surgical resection. The assay can
be used to monitor for relapse or recurrence.
[0030] Using the tumor rearrangement-specific primers to conduct
assays, one can obtain qualitative or quantitative results. The
quantitative results can be absolute amounts or relative amounts,
for example, compared to a non-rearranged sequence on the same or a
different chromosome. Assays can be conducted using the
rearrangement-specific primers and tissues or body fluids from a
subject. Suitable body fluids include whole blood, serum, and
plasma, which are collectively referred to as blood. Other body
fluids which may be used are saliva, sputum, and stool, for
example. One or more pairs of primers can be used to amplify and
assay for one or more tumor-specific rearrangements in a single
patient. Using a panel of rearrangements markers may mitigate
against any possible toss of marker during tumor growth and
progression.
[0031] The results shown below in the Examples demonstrate that
massively parallel sequencing can be used to develop personalized
biomarkers based on somatic rearrangements. We were able to
identify tumor-specific markers in each of the six breast and
colorectal cancer cases analyzed. Moreover, we demonstrated that
the identified breakpoints can be used to detect tumor DNA in the
presence of large quantities of normal DNA and in patient plasma.
These results highlight the sensitivity and specificity of the
approach and suggest broad clinical utility of the methods
disclosed here, collectively referred to as PARE.
[0032] Virtually all tumors of clinical consequence are thought to
have rearranged DNA sequences resulting from translocations and
copy number alterations and these sequences are not present in
normal human plasma or non-tumor tissues. A recent genome-wide
analysis of 24 breast cancers showed that all analyzed samples
contained at least one genomic rearrangement that could be detected
by next generation sequencing (24). From a technical perspective,
PARE-derived clinical assays should have no false positives: the
PCR amplification of aberrant fusions of DNA sequences that are
normally thousands of base pairs apart or on different chromosomes
should not occur using non-tumor DNA as a template. In contrast,
approaches that rely on monitoring of residual disease by analysis
of somatic single base alterations in specific genes are limited by
polymerase error rates at the bases of interest (25). The PCR
process generates background single base mutations that are
identical to bona, fide mutations, but does not generate
false-positive rearrangements with carefully chosen primers.
Because of the higher signal-to-noise ratio thereby obtained, PARE
theoretically permits more sensitive monitoring of tumor
burden.
[0033] The PARE approach, however, is not without limitations.
Although somatic alterations in oncogenes and tumor suppressor
genes persist throughout the clonal evolution of a tumor, it is
conceivable that some rearranged sequences could be lost during
tumor progression. The identification of several PARE biomarkers,
each specific for different chromosomal regions, would mitigate
this concern, as it is unlikely that all such markers would be lost
in any particular patient. Another limitation is the cost of
identifying a patient-specific alteration. In this prototype study,
we obtained an average of 194.7 million reads per patient,
resulting in .about.200 tags in each 3 kb bin. The current cost for
such an assay is .about.5,000, which is expensive for general
clinical use. This cost is a consequence of the high physical
coverage and the inefficiencies associated with stringent mapping
of 25 bp sequence data to the human genome. As read quality and
length continue to improve, less stringent mapping criteria and
lower physical coverage will permit analyses similar to those in
this study but with substantially less sequencing effort. Moreover,
the cost of massively parallel sequencing, which has decreased
substantially over the last two years, continues to spiral
downwards. Finally, there are clinical settings where the fraction
of any DNA from tumors, including rearranged sequences, in the
patient plasma is exceedingly small and undetectable. To be
detectable by PARE, there must be at least one rearrangement
template molecule in the plasma sample analyzed. When
disease-burden is this light, PARE may yield false negative
results. Larger studies will be needed to confirm particular
clinical uses of PARE and its prognostic capabilities.
[0034] Despite these caveats, there are numerous potential
applications of PARE. These include the more accurate
identification of surgical margins free of tumor and the analysis
of regional lymph nodes as well as the measurement of circulating
tumor DNA following surgery, radiation, or chemotherapy. Short term
monitoring of circulating tumor DNA may be particularly useful in
the testing of new drugs, as it could provide an earlier indication
of efficacy than possible through conventional diagnostic methods
such as CT scanning. Given current enthusiasm for the personalized
management of cancer patients, PARE affords a timely method for
uniquely sensitive and specific tumor monitoring.
[0035] The above disclosure generally describes the present
invention. All references disclosed herein are expressly
incorporated by reference. A more complete understanding can be
obtained by reference to the following specific examples which are
provided herein for purposes of illustration only, and are not
intended to limit the scope of the invention.
Example 1
Materials and Methods
Clinical Samples and Cell Lines
[0036] DNA samples were obtained from early passage xenografts and
cell lines of breast and colorectal cancers as described (26).
Normal DNA samples were obtained from matched normal tissue. Plasma
samples were collected from colorectal cancer patients Hx402 and
Hx403 and from an unrelated normal control. All samples were
obtained in accordance with the Health Insurance Portability and
Accountability Act (HIPAA).
Digital Karyotyping and Illumina BeadChip Arrays
[0037] A Digital Karyotyping library for colorectal cancer cell
line Co84C was constructed as previously described (6). In summary,
17 bp genomic DNA tags were generated using the NlaIII and SacI
restriction enzymes. The experimental tags obtained were
concatenated, cloned and sequenced. Previously described software
was used to extract the experimental tags from the sequencing data.
The sequences of the experimental tags were compared to the
predicted virtual tags extracted from the human genome reference
sequence. Amplifications were identified using sliding windows of
variable sizes and windows with tag density ratios .gtoreq.6 were
considered to represent amplified regions.
[0038] The Illumina Infinium II Whole Genome Genotyping Assay
employing the BeadChip platform was used to analyze the colorectal
cancer cell line Co84C at 317 k SNP loci from the Human HapMap
collection. This assay is a two step procedure; first the sample is
hybridized to a 50 nucleotide oligo, then the SNP position is
interrogated by a two-color fluorescent single base extension.
Image files and data normalization were processed as previously
described (10). Amplifications were defined as regions having at
least one SNP with a Log R ratio .gtoreq.1.4, at least one in ten
SNPs with a Log R ratio .gtoreq.1, and an average Log R ratio of
the entire region of .gtoreq.0.9. SOLiD Library Preparation and
Sequencing
[0039] Mate-pair libraries were generated for the SOLiD platform as
described (15). In brief, genomic DNA was sheared into .about.1.4
kb fragments and used as template in emulsion PCR. Fragments were
coupled to beads via an adapter sequence and clonally amplified. A
3' modification of the DNA fragments allowed for covalent
attachment to a slide. Sequencing primers hybridized to the adapter
sequence and four fluorescently labeled di-base probes were used in
ligation-based sequencing. Each nucleotide is sequenced twice in
two different ligation reactions, resulting in two base encoding
which has been shown to reduce sequencing artifacts.
[0040] Sequence data was mapped to the human genome reference
sequence (FIG. 18) using the Corona SOLiD software pipeline. All 25
bp tags (for both individual tag and mate-paired tag analyses) were
required to match the reference genome uniquely and without
mismatches.
Analysis of Single Tags for Copy Number Alterations
[0041] The SOLiD tags were filtered and the remaining tags were
grouped by genomic position in non-overlapping 3 kb bins. A tag
density ratio was calculated for each bin by dividing the number of
tags observed in the bin by the average number of tags expected to
be in each bin (based on the total number of tags obtained for
chromosomes 1-22 for each library divided by 849,434 total bins).
The tag density ratio thereby allowed a normalized comparison
between libraries containing different numbers of total tags. A
control group of SOLiD libraries made from the four matched normal
samples from Table 1 and two itional normal samples (CEPH sample
NA07357 and NA18507 used to define areas of germline copy number
variation or which contained a large fraction of repeated or low
complexity sequences. Any bin where at least 2 of the normal
libraries had a tag density ratio of <0.25 or >1.75 was
removed from further analysis.
[0042] Homozygous deletions were identified as three or more
consecutive bins with tag ratios <0.25 and at least one bin with
a tag ratio <0.005. Amplifications were identified as three or
more consecutive bins with tag ratios >2.5 and at least one bin
with a tag ratio >6. Single copy gains and losses were
identified through visual inspection of tag density data for each
sample.
Analysis of Mate-Paired Tags
[0043] Mate-paired tags mapping the reference genome uniquely and
without mismatches were analyzed for aberrant mate-pair spacing,
orientation and ordering and categorized in 13 three letter data
formats (27). Mate pairs from the same chromosome that map at
appropriate distances (.about.1 kb) and in the appropriate
orientation and ordering are categorized as AAA. Mate pairs mapping
to different chromosomes are categorized as C**. For the analysis
of translocations of the PARE approach, we focused on C** mate
pairs, while for analysis of rearrangements adjacent to copy number
alterations, we chose all non-AAA (including C**) mate pairs for
further analysis.
PARE Identification and Confirmation of Candidate
Rearrangements
[0044] To identify candidate translocations, we grouped C** mate
pair tags in 1 kb bins and looked for bin-pairs which were observed
.gtoreq.5 times in the tumor sample but which were not observed in
matched normal sample. For identification of candidate
rearrangements associated with copy number alterations, we analyzed
the 10 kb boundary regions of amplifications, homozygous deletions,
or lower copy gains and losses for neighboring non-AAA tags
observed >2 times in the tumor but not matched normal sample. In
the case of Hx402 and Hx403 the analysis of rearrangements adjacent
to copy number alterations was performed in the absence of SOLiD
libraries from normal tissue.
[0045] Mate pair tag sequences associated with a candidate
rearrangement were used as target sequences for primer design using
with Primer3 (28). When primers could not be designed from tag
sequences alone, adjacent genomic sequence up to 100 bp was used
for primer design. Importantly, the observed rearranged tag
ordering and orientation was used for Primer3 queries. Primers were
used for PCR on tumor and matched normal samples as previously
described (26). The candidate rearrangement was confirmed if a PCR
product of the expected size was seen in the tumor, but not the
matched normal sample. Sanger sequencing of PCR products was used
to identify sequence breakpoint in a subset of cases.
Detection of PARE Biomarker in Human Plasma.
[0046] To determine the sensitivity of rearranged biomarkers in the
presence of normal DNA, serial dilutions of tumor:normal DNA
mixtures were used as templates for PCR using primers for the
chromosome 4/8 translocation in Hx402. The tumor DNA dilution began
at 1:125 tumor:normal and continued as a one-in-five serial
dilution until reaching 1:390,625 tumor:normal mixture, PCR was
performed for each of the six tumor:normal DNA mixtures and for the
normal DNA control, using translocation specific primers as well as
control primers from chromosome 3.
[0047] One ml of human plasma samples were Obtained from patients
Hx402 and Hx403 and from a control individual and DNA was purified
as described (29). Whole genome amplification of plasma DNA was
performed by ligation of adaptor sequences and PCR amplification
with universal primers from the Illumina Genomic DNA Sample Prep
Kit.
[0048] Primers designed to amplify <200 bp fragments spanning
each PARE rearrangement were used in PCR from total plasma DNA
using patient or control samples. Digital PCR of plasma DNA
dilutions from patient Hx402 using rearrangement specific and
control primers were used to quantitate the fraction mutated DNA
molecules.
Example 2
Description of the Approach
[0049] The PARE approach, shown schematically in FIG. 1, in one
embodiment employs the identification of patient-specific
rearrangements in tumor samples. To determine the feasibility of
identifying such alterations using next generation sequencing
approaches, we initially analyzed four tumor samples (two colon and
two breast tumors) and their matched normal tissue samples using
the Applied Biosystems SOLiD System (Table 1). Genomic DNA from
each sample was purified, sheared and used to generate libraries
with mate-paired tags .about.1.4 kb apart. Libraries were digitally
amplified by emulsion polymerase chain reaction (PCR) on magnetic
beads (21) and 25 bp mate-paired tags were sequenced using the
sequencing-by-ligation approach (15, 22). An average of 198.1
million 25 bp reads were obtained for each sample where each read
aligned perfectly and was uniquely localized in the reference human
genome (hg18), resulting in 4.95 Gb mappable sequence per sample.
An average of 40 million mate-paired reads where both tags were
perfectly mapped to the reference human genome were obtained for
each sample. The total amount of genome base-pairs covered by the
mate-paired analysis (i.e. distance between mate-paired tags x
number of mate-paired tags) was 53.6 Gb per sample, or a 18-fold
physical coverage of the human genome.
TABLE-US-00001 TABLE 1 Summary of mate-paired tag libraries Single
tag analyses Mate-paired tag analyses Number of tags Expected
Number of mate- Distance Total physical Expected Number of matching
Total bases coverage paired tags matching between mate- coverage by
mate- genome Samples beads* human genome sequenced (bp) per 3 kb
bin human genome paired tags (bp) paired tags (bp) coverage Colon
Cancer Co108 tumor 526,209,780 121,527,707 3,038,192,675 122
21,899,809 1,371 30,024,693,714 10.0 Co108 normal 328,599,033
86,032,253 2,150,806,325 86 11,694,361 1,254 14,665,530,804 4.9
Co84 tumor 677,137,128 256,065,437 6,401,635,925 256 58,678,410
1,488 87,292,060,006 29.1 Co84 normal 486,663,520 218,280,146
5,457,003,650 218 59,019,031 1,384 81,690,396,379 27.2 Hx402 tumor
523,745,015 198,342,749 4,958,568,725 198 43,457,431 1,629
70,789,547,653 23.6 Hx403 tumor 475,658,760 164,061,938
4,101,548,450 164 37,123,395 1,705 63,295,388,475 21.1 Breast
cancer B7 tumor 840,979,999 281,027,274 7,025,681,850 281
27,548,989 1,220 33,604,662,404 11.2 B7 normal 705,704,265
253,482,262 6,337,056,550 253 57,878,644 1,404 81,271,654,770 27.1
B5 tumor 444,249,217 147,612,941 3,690,323,525 148 29,961,045 1,193
35,730,144,651 11.9 B5 normal 549,237,156 220,669,795 5,516,744,875
221 53,611,974 1,205 64,591,276,025 21.5 *Number of beads
corresponds to the number of magnetic beads containing clonally
amplified DNA fragments and represents the maximal number of raw
sequnece reads for each run.
Example 3
Identification of Somatic Rearrangements
[0050] Two methods were used to identify somatic rearrangements
from these data (FIG. 5). The first approach involved searching for
tags whose mate-pairs were derived from different chromosomes
(interchromosomal rearrangements). The high physical coverage of
breakpoints provided by the .about.40 million mate-paired sequences
per sample (Table 1) suggested that a large fraction of such
translocations could be identified. End sequences from such
mate-paired tags were grouped into 1 kb bins and those bin pairs
that were observed at least 5 times were analyzed further. The
requirement for .gtoreq.5 occurrences minimized the chance that the
presumptive fusion sequences represent incorrect mapping to the
reference genome or artifacts of library construction. Comparison
with SOLiD libraries made from the matched normal samples reduced
the possibility that the fusion sequences represented rare germline
variants rather than somatic events.
[0051] The second approach combined mate-paired tag data with copy
number alterations identified by analyses of individual 25 bp tags.
Tumor-specific copy number alterations are often associated with de
novo rearrangements (23) and the boundaries of such alterations
would be expected to contain novel junctions not present in the
human genome. To identify somatic copy number gains, losses,
high-amplitude amplifications and homozygous deletions, tags were
grouped into non-overlapping 3 kb bins. Normalized tag densities,
defined as the number of tags per bin divided by average number of
tags per bin, were determined for all 3 kb bins in each sample.
Bins that displayed tag density ratios >1.75 or <0.25 in two
or more normal tissue samples (corresponding to <6% of all bins)
were discarded from the analysis. This eliminated confounding
regions of common germline copy number variation and resulted in
892,567 bins that were analyzed in each tumor sample. Comparison of
256 million reads from colorectal tumor sample Co84 with Illumina
arrays containing .about.1 million SNP probes and with a .about.1
million Digital Karyotyping (DK) tag library Obtained with Sanger
sequencing showed high concordance for copy number alterations
among the three platforms (FIG. 6 and Table S1). With the higher
resolution afforded by the SOLiD data, we were able to identify
additional copy number changes not detected with the other methods
(Table S2). Boundary regions of copy number alteration were
analyzed to identify mate-paired tags corresponding to rearranged
DNA sequences. These included fusion of DNA sequences that have
inappropriate spacing, order or orientation on the same chromosome
(intrachromosomal rearrangements) or inappropriate joining of
sequences from different chromosomes (interchromosomal
rearrangements).
TABLE-US-00002 TABLE S1 Comparison of SOLiD sequencing, Illumina
SNP arrays, and Digital Karyotyping for analysis of copy number
alterations Digital Karyotyping Illumina SNP Arrays Tumor Left
Right Tag Density Left Right Sample Chr Boundary Boundary Size (bp)
Ratio* Boundary Boundary Amplification Co84C 6 41,273,307
43,008,812 1,735,506 9.1 41,419,345 42,485,546 Amplification Co84C
8 127,618,526 128,009,287 390,762 19.2 127,621,008 127,995,012
Amplification Co84C 8 128,750,189 128,857,861 107,673 8.3
128,750,181 128,848,183 Amplification Co84C 8 129,473,672
129,667,129 193,458 13.8 129,472,209 129,677,099 Amplification
Co84C 11 34,337,207 35,266,401 929,195 33.0 34,359,268 35,265,359
Amplification Co84C 13 109,096,557 109,553,930 457,374 9.2
109,108,212 109,557,712 Amplification Co84C 15 88,545,070
89,258,106 713,037 26.2 88,561,995 89,253,599 Amplification Co84C
19 34,570,450 34,641,949 71,500 7.9 34,561,976 34,641,548
Amplification Co84C 19 34,956,853 35,344,522 387,670 14.3
34,966,463 35,321,409 Amplification Co84C 19 36,274,262 36,388,331
114,070 6.2 36,281,540 36,385,232 Amplification Co84C 19 54,500,237
54,643,655 143,419 8.4 54,520,709 54,622,533 Illumina SNP Arrays
SOLiD sequencing Tumor Log R Left Right Tag Density Sample Chr Size
(bp) Ratio* Boundary Boundary Size (bp) Ratio* Amplification Co84C
6 1,066,202 1.9 41,418,000 42,537,000 1,119,001 16.4 Amplification
Co84C 8 374,005 2.7 127,617,000 128,010,000 393,001 150.0
Amplification Co84C 8 98,003 2.0 128,748,000 128,859,000 111,001
43.1 Amplification Co84C 8 204,891 3.4 129,471,000 129,678,000
207,001 116.6 Amplification Co84C 11 906,092 3.0 34,338,000
35,268,000 930,001 91.2 Amplification Co84C 13 449,501 2.3
109,107,000 109,557,000 450,001 33.6 Amplification Co84C 15 691,605
3.6 88,542,000 88,953,000 411,001 93.2 88,983,000 89,118,000
135,001 32.8 89,133,000 89,166,000 33,001 84.8 89,208,000
89,256,000 48,001 50.3 Amplification Co84C 19 79,573 2.2 34,548,000
34,641,000 93,001 33.9 Amplification Co84C 19 354,947 2.6
34,956,000 35,346,000 390,001 36.8 Amplification Co84C 19 103,693
2.5 36,273,000 36,396,000 123,001 21.2 Amplification Co84C 19
101,825 2.1 54,498,000 54,636,000 138,001 41.8 *Values for Tag
Density Ratios and Log R Ratios represent observed maximum values
for amplifications.
TABLE-US-00003 TABLE S2 Putative copy number alterations identified
by SOLiD sequencing in Co84 that were not identified by Illumina
SNP arrays or Digital Karyotyping Tag Density Alteration Type
Chromosome Left Boundary Right Boundary Size (bp) Ratio* Homozygous
deletion 1 83,388,000 83,532,000 144,001 0.0 Amplification 1
151,188,000 151,194,000 6,001 11.2 Amplification 1 159,393,000
159,414,000 21,001 9.7 Amplification 1 172,101,000 172,107,000
6,001 18.1 Amplification 1 179,910,000 179,916,000 6,001 17.4
Amplification 1 200,238,000 200,256,000 18,001 9.6 Amplification 1
204,188,000 204,186,000 18,001 13.2 Homozygous deletion 4 9,804,000
9,813,000 9,001 0.0 Homozygous deletion 4 69,066,000 69,171,000
105,001 0.0 Homozygous deletion 4 147,138,000 147,147,000 9,001 0.0
Amplification 5 31,749,000 31,755,000 6,001 12.3 Homozygous
deletion 5 114,279,000 114,288,000 9,001 0.0 Homozygous deletion 7
38,358,000 38,364,000 6,001 0.0 Amplification 8 145,898,000
145,725,000 27,001 11.5 Homozygous deletion 10 66,978,000
66,984,000 6,001 0.0 Homozygous deletion 13 108,681,000 108,687,000
6,001 0.0 Amplification 13 110,139,000 110,157,000 18,001 22.5
Homozygous deletion 16 54,357,000 54,378,000 21,001 0.0 Homozygous
deletion 16 59,112,000 59,130,000 18,001 0.0 Amplification 17
76,467,000 76,482,000 15,001 17.8 Homozygous deletion 18 14,268,000
14,289,000 21,001 0.0 Amplification 19 50,271,000 50,277,000 6,001
9.3 Amplification 20 25,404,000 25,428,000 24,001 13.1 Homozygous
deletion X 49,050,000 49,059,000 9,001 0.0 Homozygous deletion X
121,650,000 121,734,000 84,001 0.0 *Values for Tag Density Ratios
represent observed maximum values for amplifications.
[0052] Through these two approaches, we identified 57 regions
containing putative somatic rearrangements, with an average of 14
rearrangements per sample (Table 2). Of these, an average of seven
represented interchromosomal rearrangements and seven represented
intrachromosomal rearrangements. For confirmation, we designed
primers to 42 of the paired-end regions and used them for PCR
spanning the putative breakpoints. Thirty-five of these (83%)
yielded PCR products of the expected size in the tumor samples but
not in the normal samples (FIG. 2A-2B, Table S3). Sanger sequencing
of seven PCR products confirmed the rearrangements in all cases
tested. Though there was variation in the number of detected
alterations per sample (range 7 to 21), all four tumor samples were
found to have at least 4 bona fide somatic rearrangements through
this approach.
TABLE-US-00004 TABLE 2 Summary of rearrangements idenitified in
tumor samples Rearrange- ment type Confirmed Intra- Inter- Total
Tested somatic chromo- chromo- rearrange- rearrange- rearrange-
Sample somal somal ments ments ments Tumor and normal libraries B5
7 4 11 7 5 (71%) B7 17 4 21 16 15 (94%) Co84 0 7 7 6 4 (67%) Co108
6 12 18 13 11 (85%) Tumor libraries Hx402 7 2 9 9 4 (44%) Hx403 17
0 17 12 7 (58%)
TABLE-US-00005 TABLE S3 Confirmed somatic rearrangements in breast
and colorectal cancer samples* Forward tag Reverse tag Chrom-
Chrom- o- o- Sample some Position some Position Type Primer 1
Primer 2 B5C 3 52,638,626 3 52,573,088 AAC
AAGTTTTTCAAGCTTTACCTGAAGT TATATTGGAAGAATAGAAATGAATGG B5C 4
93,109,700 4 -93,105,085 BAC AGCCAAGTGCAATTCTCCAG
GCACACTGTTTGCAGGAATG B5C 11 57,713,780 8 -48,889,516 C**
GCCACCTTTCTTTCTTTCTGA AAGCTTTGTTTGGTTGTTCTCA B5C 18 19,141,985 20
-29,591,944 C** TGGCTTTCAAAACCCACTG TCCTTTCTGCCCATTAGGG B5C 22
48,743.603 2 -104,047,142 C** TCATGGTTTATCCACGGTGT
CACACCGCATTCACACAAAC B7C 1 -96,237,189 7 65,542,257 C**
TCAAAACAGAAAGCATTAGGC CGCATCCAAAGTATTAATAGCAA B7C 2 197,428,606 2
113,761,988 AAC AACTCCTCCCACCTCAAAATC CCAAATTGCCTGCTTAAGAGAT B7C 2
-32,084,286 3 185,241,029 C** TGCTACCAATACTTCCCACTTG
TACCGTCCTCCAGGCATGT B7C 2 114,604,628 18 53,562,784 C**
GGAGAAAACCCTGGTTATTTTTA TCCCTCATCAGAGCAAATCA B7C 3 -115,579,348 3
-115,651,310 AAC AAATTGGGAAGGATCATACTGAC TCTGAACATGCCTGATCTCATC B7C
4 785,983 4 733,804 AAC CTGAACTCCTGGGCTGAA TTGCTAAGTGATGCTACCTGTG
B7C 5 107,405,959 5 107,231,803 AAC CCTGGCCCCTTAGGTAAGAT
TGAAGAATCCTTCTAGTGATGGAA B7C 5 38,284,430 10 -44,715,202 C**
TGCAGCTTTTCTCTGTCTTCA CTGCCAGTCCAAACTGGTG B7C 6 106,401,376 6
90,853,847 AAC TGCTGTTTCAAATTCCTACAGTC TGAAATTAGGACCTGGAGCAC B7C 6
101,933,981 6 102,444,426 ABC GCCAGGTAACATGCTCACTTT
GATGCAGGAAGTTGACAGCA B7C 9 22,003,033 9 21,761,298 AAC
GGGCTAAGCTTAAGAGTCTGG GCCATGTGCAAGTCAAGAAG B7C 11 -6,436,033 11
-6,519,897 AAC TCTGCCGGCATACTGGAC TAAGGGCGATGTGAACAAGG B7C 12
65,950,588 12 65,923,399 AAC GCCCTATTTTCAGAGAAAGTGGTA
AACATCTCTTCCTTTTGAAGATCC B7C 13 60,438,525 13 52,159,979 AAC
AATTTGCTCTCATCGTATTGTGT AGCTGAATCAAAATTTCCAATG B7C X 31,583,118 X
31,179,704 AAC CTGAATCTCTTTCCAGCAAAAT AATGGGTTAAGCAGTTTAGGG Co108C
2 191,184,628 5 -104,930,827 C** TAGCATGCACCACTTTAGGC
AAAGGTTAAAGGACTGTTTTAAGTTG Co108C 2 78,849,963 6 -13,299,323 C**
GGTTCTGGAGGGTTGGAGA GTTAAGATCAACATTTTTGTTTCAAG Co108C 2 -7,268,710
6 13,299,385 C** TATGCCACCATCGCTTAGGT TCCCAGTGCAATAAAACCAA Co108C 2
-141,266,018 13 96,916,170 C** GGTGTTCTCTCTCCCATACCA
CGATCTATACACCACCCCACA Co108C 3 -60,400,269 3 -60,437,489 AAC
TGCTTTTAGTTTTGGGTACGG GCTGATTTGTTTATACCCAGTGC Co108C 3 -60,365,933
3 -60,498,861 AAC ATCCTCGGACTGGACTGAGA AACCCCATCCTGAAGCTACC Co108C
3 60,573,034 3 60,472,593 AAC GGGTTATCTCAAAAGGGCAGA
GCTCTCAATTTGTGTGATTTGG Co108C 4 81,934,151 15 54,039,041 C**
TGTGTTCCTCTCCTCTTAAGCAT GACTACAAATGGCCCAGACTC Co108C 6 -13,299,291
5 157,523,537 C** ATCCCCACATTCCCAACC CCCAGCCATATGTTGGTTTA Co108C 6
13,299,271 2 -20,956,947 C** GTATTTGTTCATGTTTGTTAGGTGTT
TCAATGGGGGAGAGAGAGC Co108C 13 34,581,537 10 67,756,452 C**
ACGTGTGTATTGGGGGTAGC CCAGATGGCTGGGTTAAATAAA Co84C 8 128,442,121 19
49,144,200 C** AGCTAGGTGGAGAATTTGTCG GGCTTCTGTAGAGTGCACATGA Co84C
11 34,790,251 13 109,267,462 C** AAGGAGATTGGTTATTGTGGAAA
CTGCAGGAACTGTCTCATTCTT Co84C 11 -34,405,644 15 88,736,701 C**
TGCTGAATCATTCTCCCAACT TGGTGATTCCACTGAGGTGA co84c 15 -89,096,347 8
127,747,412 C** GCATTCTAAAGATGAAGTCCCATT GGAAACCGTTAGTGGAAAAGTC
Hx402x 8 96,971,644 4 156,043,548 C** CAGGTGATATACCAAAGAAAATTAGG
TTTGGGTTCAGTTCTATTTGAAGA Hx402x 5 -100,413,406 5 -137,521,052 AAC
AGTCAACGCCCTAGCATGG TGGGCATGAGCAAGATATTC Hx402x 8 -144,771,376 8
-144,787,051 AAC AATCACGTTGGGTGACTGTG GTGACAGGCTGGGTGTCC Hx402x 14
-85,526,541 14 -85,560,400 AAC TGAAGGTTGAGTTGCCAGTG
TGTATGAAACATTGTAGAGGCTGT Hx403x 1 119,547,240 1 -119,550,445 BBC
AGGAGGAAAGCAACACATAGAG GGTGATTTTCAATGCATATTTCA Hx403x 5 -27,160,637
5 27,150,736 BBC AATTACCACAACTCCCAGCAG CAAAAGATTTCCAAATGCAGGT
Hx403x 11 66,674,459 11 66,662,814 AAC TGAATCAGAAAGTCTGGCAGT
CACTTGAGAATCAATGATATGCAG Hx403x 16 6,343,641 16 6,727,736 AAC
CCTAGCCCTTTGTTCCCTGT TTTGTGTACCTAGACATTCATCCAA Hx403x 16 6,574,321
16 6,759,729 AAC GCAGAGAACAGCAGAAAAGTTG AGCCAAGATCAAGCCACAGA Hx403x
16 26,579,136 16 -26,582,595 BBC TTCTCTTTCTCTGCCTTCAGTG
TTGATGATTTAGAAACTCTAGCCTGT Hx403x 17 34,622,352 17 -34,624,284 BBA
GGCTCCCCTCTCCATTCC CTGCTGACGTGCTGGTCTT *A single representative
mate pair is shown for each rearrangement. Forward and reverse tags
and their genomic coordinates correspond to F3 and R3 SOLID mate
pair tags. The type of rearrangement corresponds to the categories
described in
http://www3.appliedbiosystems.com/cms/groups/mcb_marketing/documents/gene-
raldocuments/cms_058717.pdf. AAC corresponds to mate pairs spanning
deletions; codes starting with B denote incorrect strand
orientation; codes containing a B at the middle position denote
incorrect ordering; and C** corresponds to interchromosomal
translocations. Primers 1 and 2 correspond to primers used for
confirming tumor-specific rearranged sequences.
[0053] Further examination revealed that rearrangements could be
readily identified with high confidence even in the absence of data
from matched normal DNA by using the copy number and mate-pair
coupled approach. Elimination of analysis of the matched normal
would reduce the cost and simplify the identification of
rearrangements. To test this strategy, two additional tumor samples
(Hx402 and Hx403) were then analyzed through the SOLiD approach,
but without generation of matching normal DNA libraries. We found
that it was possible to identify putative rearrangements resulting
in inter- and intra-chromosomal rearrangements at the border of
copy number variations with high specificity even in the absence of
a matched normal library. We were able to identify 11 confirmed
somatic alterations (4 and 7 in Hx402 and Hx403, respectively) out
of 21 candidate changes tested (Table S3).
Example 4
Development of PARE Biomarkers from Rearranged Sequences
[0054] Each of the rearranged sequences identified through PARE was
unique, as no identical rearrangement was found in any of the other
five tumor samples. To determine the utility of these rearranged
sequences to serve as potential biomarkers, we designed PCR assays
to detect them in the presence of increasing amounts of normal DNA.
These conditions simulate detection of tumor DNA from patient blood
or other bodily fluids where tumor DNA comprises a minority of
total DNA. PCR products representing a rearranged region from each
of the six dilutions of tumor DNA could be identified, even in
mixtures of DNA containing 1 cancer genome equivalent among
.about.390,000 normal genome equivalents (FIG. 3). Furthermore, no
background PCR products were discernable when DNA from normal
tissues was used as control.
[0055] To determine whether the rearranged sequences could actually
be detected in clinical samples, we evaluated circulating DNA from
plasma samples of patients Hx402 and Hx403. The sample from patient
Hx403 was obtained prior to surgery while the samples from patient
Hx402 were obtained prior to and after surgery. A chromosome 4:8
translocation associated with an amplification was used in tumor
Hx402 and an intra-chromosomal rearrangement associated with a
homozygous deletion of chromosome 16 was used in tumor Hx403. PCR
amplification of plasma DNA using primers spanning the breakpoints
produced products of the expected sizes only in the plasma samples
from patients with disease and not in plasma from healthy controls
(FIG. 4A). Sequencing of the PCR products from plasma DNA
identified the identical breakpoints observed in the tumor DNA
samples.
Example 5
Detection of PARE Biomarker in Human Plasma
[0056] To determine the sensitivity of rearranged biomarkers in the
presence of normal DNA, serial dilutions of tumor:normal DNA
mixtures were used as templates for PCR using primers for the
chromosome 4/8 translocation in Hx402. The tumor DNA dilution began
at 1:125 tumor:normal and continued as a one-in-five serial
dilution until reaching 1:390,625 tumor:normal mixture. PCR was
performed for each of the six tumor:normal DNA mixtures and for the
normal DNA control, using translocation specific primers as well as
control primers from chromosome 3.
[0057] One ml of human plasma samples were obtained from patients
Hx402 and Hx403 and from a control individual and DNA was purified
as described (29). Whole genome amplification of plasma DNA was
performed by ligation of adaptor sequences and PCR amplification
with universal printers from the Illumina Genomic DNA Sample Prep
Kit.
[0058] Primers designed to amplify <200 bp fragments spanning
each PARE rearrangement were used in PCR from total plasma DNA
using patient or control samples. Digital PCR of plasma DNA
dilutions from patient Hx402 using rearrangement specific and
control primers were used to quantitate the fraction mutated DNA
molecules.
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Sequence CWU 1
1
92125DNAArtificial Sequenceprimers for human genome 1aagtttttca
agctttacct gaagt 25220DNAArtificial Sequenceprimers for human
genome 2agccaagtgc aattctccag 20321DNAArtificial Sequenceprimers
for human genome 3gccacctttc tttctttctg a 21419DNAArtificial
Sequenceprimers for human genome 4tggctttcaa aacccactg
19520DNAArtificial Sequenceprimers for human genome 5tcatggttta
tccacggtgt 20621DNAArtificial Sequenceprimers for human genome
6tcaaaacaga aagcattagg c 21721DNAArtificial Sequenceprimers for
human genome 7aactcctccc acctcaaaat c 21822DNAArtificial
Sequenceprimers for human genome 8tgctaccaat acttcccact tg
22923DNAArtificial Sequenceprimers for human genome 9ggagaaaacc
ctggttattt tta 231023DNAArtificial Sequenceprimers for human genome
10aaattgggaa ggatcatact gac 231118DNAArtificial Sequenceprimers for
human genome 11ctgaactcct gggctgaa 181220DNAArtificial
Sequenceprimers for human genome 12cctggcccct taggtaagat
201321DNAArtificial Sequenceprimers for human genome 13tgcagctttt
ctctgtcttc a 211423DNAArtificial Sequenceprimers for human genome
14tgctgtttca aattcctaca gtc 231521DNAArtificial Sequenceprimers for
human genome 15gccaggtaac atgctcactt t 211621DNAArtificial
Sequenceprimers for human genome 16gggctaagct taagagtctg g
211718DNAArtificial Sequenceprimers for human genome 17tctgccggca
tactggac 181824DNAArtificial Sequenceprimers for human genome
18gccctatttt cagagaaagt ggta 241923DNAArtificial Sequenceprimers
for human genome 19aatttgctct catcgtattg tgt 232022DNAArtificial
Sequenceprimers for human genome 20ctgaatctct ttccagcaaa at
222120DNAArtificial Sequenceprimers for human genome 21tagcatgcac
cactttaggc 202219DNAArtificial Sequenceprimers for human genome
22ggttctggag ggttggaga 192320DNAArtificial Sequenceprimers for
human genome 23tatgccacca tcgcttaggt 202421DNAArtificial
Sequenceprimers for human genome 24ggtgttctct ctcccatacc a
212521DNAArtificial Sequenceprimers for human genome 25tgcttttagt
tttgggtacg g 212620DNAArtificial Sequenceprimers for human genome
26atcctcggac tggactgaga 202721DNAArtificial Sequenceprimers for
human genome 27gggttatctc aaaagggcag a 212823DNAArtificial
Sequenceprimers for human genome 28tgtgttcctc tcctcttaag cat
232918DNAArtificial Sequenceprimers for human genome 29atccccacat
tcccaacc 183026DNAArtificial Sequenceprimers for human genome
30gtatttgttc atgtttgtta ggtgtt 263120DNAArtificial Sequenceprimers
for human genome 31acgtgtgtat tgggggtagc 203221DNAArtificial
Sequenceprimers for human genome 32agctaggtgg agaatttgtc g
213323DNAArtificial Sequenceprimers for human genome 33aaggagattg
gttattgtgg aaa 233421DNAArtificial Sequenceprimers for human genome
34tgctgaatca ttctcccaac t 213524DNAArtificial Sequenceprimers for
human genome 35gcattctaaa gatgaagtcc catt 243626DNAArtificial
Sequenceprimers for human genome 36caggtgatat accaaagaaa attagg
263719DNAArtificial Sequenceprimers for human genome 37agtcaacgcc
ctagcatgg 193820DNAArtificial Sequenceprimers for human genome
38aatcacgttg ggtgactgtg 203920DNAArtificial Sequenceprimers for
human genome 39tgaaggttga gttgccagtg 204022DNAArtificial
Sequenceprimers for human genome 40aggaggaaag caacacatag ag
224121DNAArtificial Sequenceprimers for human genome 41aattaccaca
actcccagca g 214221DNAArtificial Sequenceprimers for human genome
42tgaatcagaa agtctggcag t 214320DNAArtificial Sequenceprimers for
human genome 43cctagccctt tgttccctgt 204422DNAArtificial
Sequenceprimers for human genome 44gcagagaaca gcagaaaagt tg
224522DNAArtificial Sequenceprimers for human genome 45ttctctttct
ctgccttcag tg 224618DNAArtificial Sequenceprimers for human genome
46ggctcccctc tccattcc 184726DNAArtificial Sequenceprimers for human
genome 47tatattggaa gaatagaaat gaatgg 264820DNAArtificial
Sequenceprimers for human genome 48gcacactgtt tgcaggaatg
204922DNAArtificial Sequenceprimers for human genome 49aagctttgtt
tggttgttct ca 225019DNAArtificial Sequenceprimers for human genome
50tcctttctgc ccattaggg 195120DNAArtificial Sequenceprimers for
human genome 51cacaccgcat tcacacaaac 205223DNAArtificial
Sequenceprimers for human genome 52cgcatccaaa gtattaatag caa
235322DNAArtificial Sequenceprimers for human genome 53ccaaattgcc
tgcttaagag at 225419DNAArtificial Sequenceprimers for human genome
54taccgtcctc caggcatgt 195520DNAArtificial Sequenceprimers for
human genome 55tccctcatca gagcaaatca 205622DNAArtificial
Sequenceprimers for human genome 56tctgaacatg cctgatctca tc
225722DNAArtificial Sequenceprimers for human genome 57ttgctaagtg
atgctacctg tg 225824DNAArtificial Sequenceprimers for human genome
58tgaagaatcc ttctagtgat ggaa 245919DNAArtificial Sequenceprimers
for human genome 59ctgccagtcc aaactggtg 196021DNAArtificial
Sequenceprimers for human genome 60tgaaattagg acctggagca c
216120DNAArtificial Sequenceprimers for human genome 61gatgcaggaa
gttgacagca 206220DNAArtificial Sequenceprimers for human genome
62gccatgtgca agtcaagaag 206320DNAArtificial Sequenceprimers for
human genome 63taagggcgat gtgaacaagg 206424DNAArtificial
Sequenceprimers for human genome 64aacatctctt ccttttgaag atcc
246522DNAArtificial Sequenceprimers for human genome 65agctgaatca
aaatttccaa tg 226621DNAArtificial Sequenceprimers for human genome
66aatgggttaa gcagtttagg g 216726DNAArtificial Sequenceprimers for
human genome 67aaaggttaaa ggactgtttt aagttg 266826DNAArtificial
Sequenceprimers for human genome 68gttaagatca acatttttgt ttcaag
266920DNAArtificial Sequenceprimers for human genome 69tcccagtgca
ataaaaccaa 207021DNAArtificial Sequenceprimers for human genome
70cgatctatac accaccccac a 217123DNAArtificial Sequenceprimers for
human genome 71gctgatttgt ttatacccag tgc 237220DNAArtificial
Sequenceprimers for human genome 72aaccccatcc tgaagctacc
207322DNAArtificial Sequenceprimers for human genome 73gctctcaatt
tgtgtgattt gg 227421DNAArtificial Sequenceprimers for human genome
74gactacaaat ggcccagact c 217520DNAArtificial Sequenceprimers for
human genome 75cccagccata tgttggttta 207619DNAArtificial
Sequenceprimers for human genome 76tcaatggggg agagagagc
197722DNAArtificial Sequenceprimers for human genome 77ccagatggct
gggttaaata aa 227822DNAArtificial Sequenceprimers for human genome
78ggcttctgta gagtgcacat ga 227922DNAArtificial Sequenceprimers for
human genome 79ctgcaggaac tgtctcattc tt 228020DNAArtificial
Sequenceprimers for human genome 80tggtgattcc actgaggtga
208122DNAArtificial Sequenceprimers for human genome 81ggaaaccgtt
agtggaaaag tc 228224DNAArtificial Sequenceprimers for human genome
82tttgggttca gttctatttg aaga 248320DNAArtificial Sequenceprimers
for human genome 83tgggcatgag caagatattc 208418DNAArtificial
Sequenceprimers for human genome 84gtgacaggct gggtgtcc
188524DNAArtificial Sequenceprimers for human genome 85tgtatgaaac
attgtagagg ctgt 248623DNAArtificial Sequenceprimers for human
genome 86ggtgattttc aatgcatatt tca 238722DNAArtificial
Sequenceprimers for human genome 87caaaagattt ccaaatgcag gt
228824DNAArtificial Sequenceprimers for human genome 88cacttgagaa
tcaatgatat gcag 248925DNAArtificial Sequenceprimers for human
genome 89tttgtgtacc tagacattca tccaa 259020DNAArtificial
Sequenceprimers for human genome 90agccaagatc aagccacaga
209126DNAArtificial Sequenceprimers for human genome 91ttgatgattt
agaaactcta gcctgt 269219DNAArtificial Sequenceprimers for human
genome 92ctgctgacgt gctggtctt 19
* * * * *
References