DIAGNOSTIC METHODS BASED ON SOMATICALLY ACQUIRED
REARRANGEMENT
Individualised health-care is a major goal for medicine in the next 5-1 0 years. Numerous
advances are required to attain this objective, including the development of sensitive and
specific biomarkers for measuring disease burden.
By way of one example, personalising cancer medicine depends upon the implementation of
personalised diagnostics. Since cancer is, at its core, driven by somatic mutation, detailed
genomic screening is likely to play a central role in facilitating individual therapeutic choices.
As the range of drugs and other therapies for cancer continues to increase, there is an
increasingly urgent need for sensitive and specific measures of disease burden to guide
treatment regimens.
In haematological malignancies, there is routine quantification of residual disease levels
through assays for recurrent genomic rearrangements. This has been made possible by the
discovery that leukaemias are associated with characteristic genomic rearrangements that do
not require either genome-wide screening or development of patient-specific assays. In solid
tumours, however, methods for quantifying disease burden are less sensitive and less
specific. Radiological imaging is routinely used for staging patients, but can only detect gross
lesions > 1cm in size, already representing many millions of cancer cells. Serum markers,
such as PSA for prostate cancer, can be helpful, but are not available for many tumour types
and frequently suffer from problems of non-specificity. Immunological detection of circulating
tumour cells is sensitive down to 1 cancer cell in thousands of normal cells, but only detects
cells present in the blood and can result in false positive calls from non-malignant cells
expressing the marker of interest.
Tumour cells release naked DNA into the plasma as they necrose or apoptose, and the level
of circulating free DNA correlates with disease burden. This can be used to monitor levels of
tumour-specific point mutations or epigenetic changes in oncogenes with some prognostic
value. However, the fraction of circulating naked DNA that derives from tumour cells is 0.0 1%
or lower in many cases, and current methods for discriminating a single mutated base at this
depth are inadequate. Thus, the existing strategies, which are based around point mutations
or epigenetic changes in plasma DNA, suffer poor sensitivity and lack of specificity.
The present invention addresses the issue of detection and monitoring of diseases to support
a personalized medicine approach.
Statements of invention
The present invention relates to a method suitable for monitoring a disease, the method
comprising;
a identifying a somatically acquired genomic rearrangement associated with a disease
state in a patient, wherein the identification is carried out by genome-wide analysis of the
nucleic acid of that patient, and
b monitoring the changes in levels of nucleic acid containing the genomic
rearrangement, and/or quantifying the levels of nucleic acid containing the genomic
rearrangement, as a marker for the progression or severity of a disease in that patient.
The present invention also relates to a method suitable for monitoring a disease, the method
comprising monitoring the changes in levels of nucleic acid containing a genomic
rearrangement, and/or quantifying the levels of nucleic acid containing a genomic
rearrangement, wherein the genomic rearrangement is a somatically acquired mutation
associated with a disease progression or severity in a patient, and wherein the identification
of the rearrangement has been carried out by genome-wide analysis of the nucleic acid of
that patient.
In a further aspect the invention relates to a method of medical treatment comprising,
monitoring according to the present invention and then additionally the step of treating the
patient with an appropriate therapy, or changing therapy, or stopping therapy, where
necessary, depending upon disease severity or progression, as indicated by the presence of,
or level of, nucleic acid containing the genomic rearrangement.
In a further aspect the invention relates to use of a patient specific genomic rearrangement as
a biomarker for progression of disease in that patient, wherein the genomic rearrangement is
a somatically acquired mutation.
In a further aspect the invention relates to a method for assessing the efficacy of a treatment,
the method comprising:
i identifying a somatically acquired genomic rearrangement associated with a
disease state in a patient, wherein the identification is carried out by genome-wide analysis of
the nucleic acid of that patient, and
ii monitoring the changes in levels of nucleic acid containing the genomic
rearrangement as a marker for the progression or severity of a disease in that patient in
response to a treatment, thereby assessing the efficacy of the treatment.
Figures
Figure 1 illustrates quantitative detection of genome rearrangements in plasma DNA
Figure 2 illustrates analysis of serial samples
Detailed description
The present invention generally relates to detection of rearrangements in nucleic acid that are
associated with a disease state of a patient by genome wide analysis of the nucleic acid of
that patient. Technologies now available allow the mapping of many nucleic acid breakpoints
in a tissue sample, across a genome. Once suitable nucleic acid rearrangement biomarkers
have been identified for a patient the progression of a disease may be followed by monitoring
the increase or decrease in levels of nucleic acid containing said breakpoint(s) over time in
that patient. Where the nucleic acid having the rearrangement is detectable in the blood or
plasma, then blood or plasma samples may be sampled to easily monitor the progression
disease. Treatments for the disease in a patient may be stopped once disease progression
has halted, or been reversed, or is not detectable, as assessed by rearrangement biomarker
levels. Treatment regimens may be altered or terminated if the disease burden is not
reducing, as assessed by rearrangement biomarker levels. The effects of, and thus suitability
of, different treatments can also be assessed. .
The method of the invention may also be used to determine if relapse has occurred, to allow
treatment to be restarted, if necessary.
In the example of cancer, rearrangement screens are potentially applicable to all tumour
types in which a diagnostic sample can be accessed for genomic screening. Most patients
with solid tumours undergo either biopsy or full surgical resection of their cancer during the
course of their therapy, meaning that access to tumour DNA is usually achievable. In our
experience to date, more than 99% of samples analysed , across a wide variety of tumour
types, have had at least one identifiable tumour-specific genomic rearrangement.
Thus in a first aspect the invention relates to a method for determining the progression of a
disease, the method comprising
a identifying a somatically acquired genomic rearrangement associated with a disease
state in a patient, wherein the identification is carried out by genome-wide analysis of the
nucleic acid of that patient, and
b monitoring the changes in levels of nucleic acid containing the genomic
rearrangement as a marker for the progression of a disease in that patient.
A disease, as disclosed herein, may be any disease associated with a somatic
rearrangement. Diseases may include, for example, cancers, such as solid tumours,
Paroxysmal nocturnal hemoglobinuria, Neurofibromatosis 1 and 2 , McCune-Albright,
Incontinentia pigmenti, and Proteus syndrome.
In one aspect of the invention the disease is a cancer, such as a solid tumour, e.g. breast
cancer, non-small cell lung cancer, colon cancer, pancreatic, ovarian and bone cancers.
In one aspect the disease is characterized in that nucleic acid comprising the rearrangement
is detectable in a sample from a body fluid such as blood, serum, plasma, lymph, sputum,
urine, faeces or saliva.
Rearrangements associated with disease states herein, such as cancer, are not necessarily
causative of that disease, although they may cause or contribute to the disease phenotype.
However, it is only necessary that the rearrangement associates with the disease such that
monitoring of the rearrangement can allow the progression or severity of the disease to be
followed . In one aspect the invention relates to monitoring using rearrangements that are
not causative of disease, or not solely causative of disease.
Analysis of the nucleic acid of a patient for identification of rearrangements is suitably carried
out on nucleic acid from diseased cells in the body, such as a tumour, for example a solid
tumour. In one aspect the genome wide nucleic acid analysis is carried out on biopsied or
surgically resected tumour tissue. Rearrangement screens are suitably carried out on tumour
nucleic acid derived from a population of cells. Thus in one aspect the source of nucleic acid
for genome wide analysis is a known diseased cell or tissue that allows the identification of
mutations that are associated with that disease.
In an alternative aspect of the invention nucleic acid may be taken directly from a tissue or
fluid , such as blood or plasma or serum, which is not itself known to be diseased , but from a
patient known to have a disease. Somatically acquired genomic rearrangements are also
useful as a marker of disease in such a case, where the assumption is made that the
mutation is associated with the disease state.
Nucleic acid may be obtained from cultured cells, as well as directly from body tissue or fluid.
The nucleic acid may be DNA, or RNA.
Genome wide analysis as disclosed herein is, in one aspect, analysis of all or a significant
part of the genome of an individual to identify mutations in the form of rearrangements that
are found in diseased tissue of the individual, such as a tumour. Genome wide analysis in
one aspect is the identification of rearrangement mutations from an individual that correlate
with disease by analysis of random nucleic acid fragments or regions from that individual,
suitably without use of probes or primers that are known to be specific to nucleic acid from
that individual. Thus it is not necessary to have complete coverage of a genome, although in
one aspect techniques that allow analysis of the whole genome, at least based upon a
statistical analysis of coverage, are preferred.
Somatically acquired genomic rearrangements may include deletions, inversions,
translocations and amplifications. In one aspect the rearrangement is detectable by a change
in the length of a restriction fragment within which the mutation is located , in comparison with
the patient's normal (non-mutated) genome.
In one aspect the analysis is carried out by sequencing DNA, for example, the sequencing of
randomly generated fragments of the DNA of an individual. In one aspect the sequencing is
sequencing of a library of sized DNA fragments, such as 400-500bp. In one aspect the
technique used is massively parallel sequencing , as described herein, and also in Campbell
et al Nature Genetics, Vol 40, number 6 , June 2008, page 722 - 729.
Suitable massively parallel sequencing platforms also include the SOLiD platform (Applied
Biosystems) and the use of the 454 sequencer (Roche).
In one aspect the sequencing is carried out using paired end sequencing. Suitably paired
reads from in the order of 60 million fragments are generated, which is generally sufficient to
identify >50% of somatic genomic rearrangements present in a sample.
Paired end sequencing methods are disclosed in, for example, Genome Res. 2009. 19 : 521 -
532.
In one aspect the genomic rearrangements are prioritized. Prioritisation may be by, for
example, including 1 or more of the following steps:
• >2 reads spanning the same rearrangement;
• High confidence mapping for both ends;
• Reads mapping < 100kb apart on the same chromosome;
• Both ends mapping to within 100kb of a change-point in copy number identified by the
segmentation algorithm.
In one aspect the step of monitoring the changes in levels of nucleic acid containing the
genomic rearrangement is a DNA amplification assay, such as a PCR assay, for example a
nested PCR approach. Reference herein to PCR generally refers to DNA amplification
technologies, including the polymerase chain reaction specifically. Suitably primers designed
to specifically identify the nucleic acid rearrangement are used in an amplification process.
In one aspect the PCR process is carried out on a nucleic acid sample obtained from blood ,
or serum.
In one aspect the size of the initial PCR product is less than < 200 bp, preferably less than
190 bp, 180 bp, 170 bp, 160 bp, 150 bp.
Suitably assays to monitor the level of a specific rearrangement are preferably substantially
quantitative.
Changes in levels of the nucleic acid can be made by absolute or relative measurements.
For example, the ratio of the level of 'normal' genomic DNA vs the mutated genomic DNA can
be used. Alternatively, the absolute amount of mutated DNA can be measured, for example,
DNA per ml of plasma. Measurement of the change in levels of nucleic acid, or quantification
of levels of nucleic acid, can be either by ratio or absolute concentration measurement.
In one aspect the assay of the invention for monitoring changes in levels of nucleic acid in a
patient in step is linearly quantifiable down to the level 25pg of DNA per assay.
In one aspect a one log increase in the absolute quantity of DNA detected with the
rearrangement is considered to be a significant increase in disease burden, and may require
treatment.
In one aspect of the invention multiple genomic rearrangements are monitored, to provide a
genetic fingerprint of an individual.
In a further aspect the invention relates to a method of medical treatment comprising the
monitoring of the invention, and additionally then comprising the step of treating the patient
where necessary, or changing treatment, or stopping treatment if treatment is ongoing,
depending upon disease severity or progression as indicated by the level of nucleic acid
containing the informative rearrangement.
In a further aspect the invention relates to use of a patient specific genomic rearrangement as
a biomarker for progression of disease in that patient.
It will be appreciated that the progression of a disease may be followed by monitoring the
change in an identified marker over time. The severity of a disease may be assessed by a
measurement at a single point in time of a biomarker whose presence or concentration is
indicative of disease severity. For example, the presence in the blood of a somatic DNA
rearrangement originally identified in a solid tumour may indicate the progression of a cancer
beyond a certain disease state.
Thus the invention relates to a method for determining a disease state, the method
comprising identifying a somatically acquired genomic rearrangement associated with a
disease state in a patient wherein the identification is carried out by genome-wide analysis of
the nucleic acid of that patient. Suitably the genomic rearrangement is identified in a patient
in a tissue or fluid, such as blood or plasma or serum. Preferably the genomic rearrangement
is identified at a site, or in an organ tissue or fluid, other than in the primary diseased tissue
from which the genome wide analysis was carried out.
For example, a solid tumour may represent a primary disease tissue, and the presence of a
somatically acquired genomic rearrangement in the blood or other body fluid might be
indicative of a certain disease progression of the cancer, and allow a therapeutic treatment to
be determined.
In one aspect the invention also relates to a method for determining a treatment regimen for a
disease, the method comprising quantifying the level of a somatically acquired genomic
rearrangement associated with a disease state in a patient in a tissue or fluid, preferably
other than in the primary diseased tissue, wherein the identification is carried out by genomewide
analysis of the nucleic acid of that patient, and selecting a treatment regimen based
upon the level of said genomic rearrangement.
In a further aspect the invention relates to a method for assessing the efficacy of a treatment,
the method comprising:
i Identifying a somatically acquired genomic rearrangement associated with a
disease state in a patient, wherein the identification is carried out by genome-wide analysis of
the nucleic acid of that patient, and
ii monitoring the changes in levels of nucleic acid containing the genomic
rearrangement as a marker for the progression of a disease in that patient in response to a
treatment, thereby assessing the efficacy of the treatment.
The treatment may be a novel treatment, in which case the method of the invention may not
only be used to monitor the best treatment for a patient, it can also be used to generally
determine the efficacy of new treatment regimens and new drug or other therapeutic
treatments. This application is not limited to humans, but could also be applied to animals.
The invention thus also extends to a method for assessing the efficacy of a drug or treatment
regimen, the method comprising treatment of an individual in need thereof with the drug or
treatment regimen and then monitoring the progression or severity of disease after treatment
by measurement of levels of nucleic acids with somatic rearrangements as a biomarker for
disease.
In one aspect the invention also relates to a method for monitoring cell killing, wherein the
killing of cells using a drug or treatment regimen is monitored by the release of nucleic acid
comprising a somatic rearrangement.
Cancer Res 2007; 67: ( 19). October 1, 2007 p9364 - 9370 discloses monitoring of cell killing
in an animal model.
If a patient is being treated with a drug or other therapy, for example, surgery, or radiotherapy
or chemotherapy, then the effectiveness of that treatment can be monitored by looking for
levels of the nucleic acid having the rearrangement in the patient.
Suitable treatments include the use of surgery, chemotherapy, radiotherapy, monoclonal
antibodies, hormonal therapy and molecularly targeted therapy or a combination thereof.
In addition, where a patient has been treated for a disease and is in remission, then the
continued remission status of the patient can be monitored. Should the patient come out of
remission (relapse), then treatment can be restarted. Thus monitoring the progression of
disease, as referred to herein, also includes monitoring the reoccurrence of disease after
remission, and optionally treatment of the disease after relapse. The present invention may
be used to monitor relapse after a period of, for example, hours, days, weeks or months after
remission or the last cycle of treatment.
In a preferred aspect of the invention, there is provided a method for determining the
progression of cancer, the method comprising:
a) identifying a somatically acquired genomic rearrangement in nucleic acid from a tumour
sample of a patient by genome-wide analysis through paired-end sequencing ;
b) designing a quantitative assay for identifying and measuring the somatically acquired
genomic rearrangements;
c) obtaining one of more further samples from the patient during future stages of therapy;
d) using the assays to measure levels of nucleic acids with somatic rearrangements in the
samples obtained during step (a) and step (c); and optionally
e) determining the progression and/or severity of disease for the patient by comparing the
levels of nucleic acids with somatic rearrangement measured in step (d).
In a further aspect of the invention pregnant women may be screened to determine whether
hereditable diseases have been passed on to their children. Where a father is known to have
a condition that is associated with a somatically acquired genomic rearrangement, the
presence of this rearrangement can be assessed in, for example, the blood or serum, of the
mother. Thus in one aspect the invention relates to a method suitable for monitoring a
disease, the method comprising;
a identifying a somatically acquired genomic rearrangement associated with a disease
state in a patient, wherein the identification is carried out by genome-wide analysis of the
nucleic acid of that patient, and
b monitoring the changes in levels of nucleic acid containing the genomic
rearrangement, and/or quantifying the levels of nucleic acid containing the genomic
rearrangement, in a pregnant woman as a marker for the progression or severity of a
disease in the foetus.
The teaching of all references in the present application, including patent applications and
granted patents, are herein fully incorporated by reference. Any patent application to which
this application claims priority is incorporated by reference herein in its entirety in the manner
described herein for publications and references.
For the avoidance of doubt the terms 'comprising', 'comprise' and 'comprises' herein is
intended by the inventors to be optionally substitutable with the terms 'consisting of, 'consist
of, and 'consists of, respectively, in every instance. The term "about" (or "around") in all
numerical values allows for a 5% variation, i.e. a value of about 1.25% would mean from
between 1.19%-1 .31 %.
It will be understood that particular embodiments described herein are shown by way of
illustration and not as limitations of the invention. The principal features of this invention can
be employed in various embodiments without departing from the scope of the invention.
Those skilled in the art will recognize, or be able to ascertain using no more than routine
study, numerous equivalents to the specific procedures described herein. Such equivalents
are considered to be within the scope of this invention and are covered by the claims. All
publications and patent applications mentioned in the specification are indicative of the level
of skill of those skilled in the art to which this invention pertains. All publications and patent
applications are herein incorporated by reference to the same extent as if each individual
publication or patent application was specifically and individually indicated to be incorporated
by reference.
The use of the word "a" or "an" when used in conjunction with the term "comprising" in the
claims and/or the specification may mean "one," but it is also consistent with the meaning of
"one or more," "at least one," and "one or more than one." The use of the term "or" in the
claims is used to mean "and/or" unless explicitly indicated to refer to alternatives only or the
alternatives are mutually exclusive, although the disclosure supports a definition that refers to
only alternatives and "and/or." Throughout this application, the term "about" is used to
indicate that a value includes the inherent variation of error for the measurement, the method
being employed to determine the value, or the variation that exists among the study subjects.
As used in this specification and claim(s), the words "comprising" (and any form of
comprising, such as "comprise" and "comprises"), "having" (and any form of having, such as
"have" and "has"), "including" (and any form of including, such as "includes" and "include") or
"containing" (and any form of containing, such as "contains" and "contain") are inclusive or
open-ended and do not exclude additional, unrecited elements or method steps.
The term "or combinations thereof as used herein refers to all permutations and
combinations of the listed items preceding the term. For example, "A, B, C, or combinations
thereof is intended to include at least one of: A, B, C, AB, AC, BC, or ABC, and if order is
important in a particular context, also BA, CA, CB, CBA, BCA, ACB, BAC, or CAB. Continuing
with this example, expressly included are combinations that contain repeats of one or more
item or term, such as BB, AAA, BBC, AAABCCCC, CBBAAA, CABABB, and so forth. The
skilled artisan will understand that typically there is no limit on the number of items or terms in
any combination, unless otherwise apparent from the context.
All of the compositions and/or methods disclosed and claimed herein can be made and
executed without undue experimentation in light of the present disclosure. While the
compositions and methods of this invention have been described in terms of preferred
embodiments, it will be apparent to those of skill in the art that variations may be applied to
the compositions and/or methods and in the steps or in the sequence of steps of the method
described herein without departing from the concept, spirit and scope of the invention. All
such similar substitutes and modifications apparent to those skilled in the art are deemed to
be within the spirit, scope and concept of the invention as defined by the appended claims.
The present invention is now exemplified with reference to the following examples, which are
not limiting upon the invention
Methods
The following steps are employed in this example of the invention
Identification of tumour-specific genomic rearrangements by massively parallel sequencing.
Mapping rearrangements to base-pair resolution.
Design of PCR-based assays for sensitive and specific quantification of tumour burden
Extraction of free DNA from serum and quantification of tumour load with appropriate
controls.
Identification of tumour-specific rearrangements by massively parallel sequencing
Genomic DNA is extracted from the tumour sample using phenol-chloroform extraction or
other standard protocols. Libraries are prepared from tumour DNA according to the protocol
recommended by the manufacturer of the massively parallel sequencing platform to be used.
For the Solexa sequencing platform (Genome Analyzer, lllumina, San Diego CA), genomic
DNA ^g) is randomly sheared using the nebuliser supplied with the Genome Analyzer
instrument according to the manufacturer's instructions. The fragmented DNA is end-repaired
using T4 DNA polymerase and Klenow polymerase with T4 polynucleotide kinase to
phosphorylate the 5' ends. A 3' A overhang is created using a 3'-5' exonuclease-deficient
Klenow fragment, and lllumina paired-end adapter oligonucleotides are ligated to the sticky
ends thus created. The ligation mixture is electrophoresed on an agarose gel, and sizeselected
by excising the DNA fragments of 400-500 base pairs in length. DNA is extracted
from the gel and enriched for fragments with Solexa primers on either end by a limited cycle
PCR reaction, following the manufacturer's instructions.
The Genome Analyzer paired-end flow-cell is prepared on the supplied cluster station
according to the manufacturer's protocol. Clusters of PCR colonies are then sequenced on
the Genome Analyzer platform using recommended protocols from the manufacturer. Pairedend
sequencing of at least 35bp from either end gives the optimal coverage for identifying
rearrangements, although they can be found with longer read-length, single-end reads.
Images from the instrument are processed using the manufacturer's software to generate
FASTQ sequence files.
Most of the current generation of massively parallel sequencing platforms can be used to
identify genomic rearrangements, including the SOLiD platform (Applied Biosystems) and the
454 sequencer (Roche). Longer insert sizes increase coverage, and allow greater confidence
in recognising clusters of rearrangements. Where sequencing data from the constitutional
(germline) DNA from the same patient is available, this can be used to aid the distinction
between germline and somatic rearrangements.
Sequence data are aligned to the reference human genome, using any of several freely
available packages. We use the MAQ algorithm vO.4.3 (available at
http://maq .sourceforge.net/maq-man.shtml). Reads in which the two ends failed to align to
the genome in the correct orientation and distance apart are further screened with the
SSAHA algorithm.
Removal of artefacts
Reads where the two ends map back to within 500bp of one another, but with one of the two
ends in the incorrect orientation are excluded from analysis, since they are likely to be
artefacts due to either mis-priming within the PCR colony or intra-molecular rearrangements
generated during library amplification. Reads which are exact duplicates of one another
(created during the PCR enrichment step) are identified by the fact that the two ends of the
sequences map to identical genomic locations: only the fragment with the higher mapping
quality is retained. Spurious mapping of DNA from sequence gaps in the reference genome is
reduced by excluding regions within 1Mb of a centromeric or telomeric sequence gap from
copy number and rearrangement analyses (see a list of current sequence gaps, for example,
at: http://genome.ucsc.edu/cgi-bin/hgTables).
Copy number algorithm
To correct for varying levels of uniqueness across the genome, an in silico simulation of
paired-end short reads is performed by creating paired sequences of 35 bases each end,
500bp apart (or the equivalent if different libraries have been used), with simulated pairs
located every 35bp along the genome. These simulated reads are mapped to the genome
using the MAQ algorithm. On the basis of this, the genome is divided into non-overlapping ,
unequal width windows which contain a constant number of in silico reads mapped with high
uniqueness. With the window boundaries set, the number of paired-end reads mapping
uniquely within each window is counted. This forms the raw input to a binary circular
segmentation algorithm originally developed for genomic hybridisation microarray data. This
algorithm, implemented in R as the DNAcopy library of the Bioconductor project (see
http://www.bioconductor.org/), identifies change-points in copy-number by iterative binary
segmentation. We use a = 0.01 , together with a smoothing parameter of 2 and 2 standard
deviations for pruning of probable false positives after segmentation, although the modelling
generally gives similar results for different parameter choices.
Mapping rearrangements to base-pair resolution
The following criteria are used for prioritising incorrectly mapping reads for confirmatory
screening:
>2 reads spanning the same rearrangement;
High confidence mapping for both ends;
Reads mapping < 100kb apart on the same chromosome;
Both ends mapping to within 100kb of a change-point in copy number identified by the
segmentation algorithm.
Primers are designed to span the possible breakpoint by locating them in the 1kb outside the
paired-end reads, for a maximum product size of 1kb. PCR reactions are performed on
tumour and normal genomic DNA for each set of primers. Products giving a band are
sequenced by conventional Sanger capillary methods and compared to the reference
sequence to identify breakpoints. Somatically acquired, tumour-specific rearrangements are
defined as those PCR reactions giving a convincing band in the tumour DNA with no
matching band in the normal DNA, seen in at least two separate reactions, together with
unambiguously mapping sequence data suggesting a rearrangement.
Alternatively, rearrangements can be mapped by de novo assembly across the breakpoint.
This is accomplished by extracting paired-end reads where one end maps in the location of
the breakpoint. These reads can be assembled into longer contigs, which can be aligned
against the reference genome to identify the exact location of the breaks.
Design of PCR-based assays for quantification of tumour-specific rearrangements
We have found nested PCR to be a sensitive and specific method for amplification of tumourspecific
genomic rearrangements.
Each confirmed somatic structural rearrangement is assessed for suitability as a DNA
marker:
Copy number change
It is important to select DNA markers present in the majority of the tumour cells.
Rearrangement screens are carried out on tumour DNA derived from a population of cells,
therefore the output of the sequencing experiment is a tumour cell population average of
rearrangement. We seek to use rearrangements that i) are prevalent in the sequencing data
and ii) have clear copy number changes, since these will be present in the majority (or all) of
tumour cells.
Uniqueness of surrounding DNA
Each assay must be specific for a particular rearrangement. The repetitive nature of the
genome means that non-unique, repetitive sequences are located at multiple positions across
the genome. In order to obtain highly specific assays the repetitive sequences surrounding
each breakpoint are masked using repeat masker software (www.repeatmasker.org). This
excludes a proportion of somatically acquired breakpoints from further analysis because of
closely surrounding repeats. For some breakpoint junctions, no or only short stretches of
nucleotides are masked out. This allows specific assays to be designed for these
rearrangements if repeat sequences are avoided.
Number of assays
We aim to design probes to 3-4 tumour-specific rearrangements per patient. Having multiple
assays per patient increases confidence in the final result, although it is not always possible
to identify this number of suitable rearrangements in every patient. Tumour specific assays
are run alongside 4 control assays that are designed to recognise wild-type regions of the
genome.
Assay / oligo design
We take a nested PCR approach to identify cancer-specific rearrangements from the high
background of circulating DNA derived from normal cells. Primers are designed using primer
3 (http://frodo.wi.mit.edu/) to avoid repetitive sequence and span the rearrangement
breakpoint. The size of the initial PCR product should be kept to a minimum (< 200 bp)
because circulating tumour DNA tends to be most abundant in this size range. The sequence
amplified in the 1st round of PCR is used as a template to design a dual labelled DNA probe
("taqman") style quantitative PCR assay using Beacon Designer software (Premier Biosoft
International). This program selects primers and a dual labelled [5' FAM, 3' BHQ1 ] DNA
probe. Due to the strict size constraints, overlap between the real time and 1st round primers
is sometimes required.
Extraction of DNA from plasma/serum and quantification of tumour burden
DMA extraction
Debris are removed from patient plasma or serum by centrifugation at 16 , 000 g for 10
minutes. DNA is extracted from 2-20ml_ of the resultant supernatant using the QIAamp
MinElute Virus Vacuum Kit (Qiagen). DNA is eluted in 20m I of the supplied elution buffer and
the entire volume is used as template in the following PCR.
Multiplex PCR
The entire quantity of DNA extracted from 2-20ml_ of patient plasma (or 10 fold serial dilution
derived from this) is combined with all patient specific 1st round PCR primers along with 1st
round control region primers and subjected to 20 cycles in a multiplex PCR. Combining all
primers ensures that the highest possible quantity of DNA is available to each primer set.
Real time PCR
Ten-fold dilutions were made of the nested PCR product and 5m I is used as template in
individual real time PCR reactions using rearrangement specific primers and probes.
Quantification
Using serial dilutions of the patient's tumour DNA in normal DNA (or water) allows production
of a standard curve, since the amount (in pg) of tumour DNA per reaction is known. Using the
curve of best fit applied to the standards then allows interpolation of the amount of tumour
DNA present in the known volume of plasma/serum. In our experience, the nested , real-time
PCR is capable of detecting down to 1 copy of the target rearrangement present in the whole
volume of plasma analysed.
Results
We investigated two patients with metastatic breast cancer. Massively parallel paired-end
sequencing was used to identify somatically acquired genomic rearrangements from the
genomes of both primary cancers and PCR assays were designed to amplify across multiple
rearrangements from each genome (see Figure 1 below) The PCR products were sequenced
to identify the breakpoints to base-pair resolution. We then designed nested real-time PCR
assays to amplify and quantify the amount of tumour DNA.). After confirming the success of
the PCR design on DNA from the cancer we then examined plasma samples taken at first
presentation of disease in both cases. DNA was extracted from 2ml_ of plasma and analysed
by the patient-specific real-time assays we designed . Figure 1 shows the results from the
real-time PCR reactions. The curves show the amount of fluorescence generated by the real¬
time (Taqman) probes on the y axis with the number of PCR cycles on the x axis. The dark
horizontal line around the middle of each figure 1 graph marks the level of fluorescence at
which a reaction is deemed to reach positivity, so that the earlier (more leftward) a curve
crosses the threshold, the greater the amount of target DNA in the reaction. Curves derived
from plasma DNA from a normal individual (the negative control for the reaction) and curves
showing serial 10-fold dilutions of the patient's plasma in water are identified by separate
arrows in Figure 1. The left-hand graph for each patient shows the results for tumour-specific
rearrangements. Clearly, the patient plasma samples are positive, whereas the negative
control plasma (from a normal individual) is negative throughout. The right-hand graph for
each patient shows the result for a normal region of the genome (positive control), which, as
expected, is positive in both the patient and the normal control.
Figure 1: Quantitative detection of genomic rearrangements in plasma DNA of patients with
breast cancer: somatically acquired genomic rearrangements were identified in the primary
breast cancers of two patients with metastatic disease by massively parallel sequencing.
DNA extracted from 2ml_ of plasma taken at diagnosis and serial 10-fold dilutions were
screened by nested PCR with a final round of real-time PCT Robust detection of
rearrangements ( 1 shown and 2 others not shown for each patient) was possible in both
patients. Comparison of tumour-specific and control reactions in the dilution series suggests
that in each patient the ratio of tumour-specific DNA to total DNA in plasma is 1:10 , the total
amount of plasma DNA extracted was ~ 100x greater for patient PD3722a than PD3770a.
The results show that these somatic rearrangements can be quantitatively detected in plasma
DNA. In particular, the following key features of the assay can be demonstrated from the
analyses:
• The assay is highly sensitive, since the analyses were positive even with dilution of
the plasma ( 1:10 dilution for the first patient, and 1:1000 for the second patient,
equivalent to detecting a signal in the DNA from just 2m I_ of plasma, since 2ml_ was
the starting volume).
• The assay is highly specific, since normal plasma DNA did not reveal a signal, even
with nested PCR.
• The assay is quantitative, since the dilutions of plasma showed linear increases in
the Ct , with robust separation between curves.
We have subsequently analysed serial samples from a third patient with cancer undergoing
chemotherapy (Figure 2 below). As we did for the first two patients, a genome-wide
rearrangement screen was undertaken using massively parallel sequencing to identify
somatically acquired rearrangements. Two of these were selected for assay design. Serial
dilutions of tumour DNA were made in normal DNA.
Figure 2A shows analysis of rearrangements 1 and 2 in duplicate reactions across a dilution
series of tumour DNA into normal DNA. Where Ct<27, the absolute amount of tumour DNA
can be estimated from the line of best fit . For Ct>27, disease can only be classified as
detectable or undetectable. However, the assay appears able to detect a single copy of the
rearrangement present in the reaction. Figure 2B shows the estimated amount of tumour
DNA per mL of serum from 6 samples collected at milestone time points in the patient's
clinical course.
Panel A of figure 2 shows testing of the assays in replicate experiments on the serial
dilutions. The results demonstrate that the assays are robust, reproducible and linearly
quantifiable down to about 25pg of DNA per reaction. Given that a diploid human cell
contains ~6.75pg DNA, this is equivalent to ~4 genomes per reaction. With lower amounts of
tumour DNA in the reaction (5pg and 10pg per reaction), we find that reactions are either
positive or negative (shown as points to the far right of the graph). This implies that in the
negative reactions, no copies of the rearrangement were present, whereas in the positive
reactions, 1 or 2 copies were present. A major finding therefore is that the nested real-time
PCR assay would be capable of detecting a single copy of the rearrangement present in
many millilitres of blood.
We next screened serial serum samples from the patient collected at time-points during her
chemotherapy (Figures 2B and 2C). Unfortunately, no samples were available from before
starting therapy. However, from the mid-point of her first-line chemotherapy through to the
end of second-line chemotherapy, residual disease was detectable in the serum at the limits
of detection of the assay. She unfortunately suffered clinical progression within a month or
two of completing chemotherapy, and this was associated with an increase in the levels of
disease detectable in her serum. In the time to schedule salvage chemotherapy, the levels of
disease increased further.
These serial analyses demonstrate that the assay is capable of detecting disease even when
present in minimal amounts clinically, and that the quantification of disease burden correlates
with disease progression.
Future Studies
Aims:
We intend to measure the prognostic significance of tumour-specific rearrangements
quantified in plasma DNA for:
100 patients with non-metastatic breast cancer treated in an adjuvant therapy setting;
100 patients with stage II I or advanced stage I I colorectal carcinoma.
Non-metastatic breast cancer
Breast cancer is responsible for 16% of cancer deaths in women in the UK. For patients
without known distal metastases at diagnosis, treatment is generally delivered with curative
intent, but relapse rates range between 20-40% within 5 years depending on localised nodal
involvement, size of the primary tumour and oestrogen receptor status. Many questions
remain unanswered regarding the best use of adjuvant therapy in this clinical setting, and an
accurate method to quantify disease burden would be invaluable for establishing
personalised treatment regimens and optimising treatment intensity and duration.
Stage III and high-risk stage II colorectal cancer
Colorectal cancer is responsible for 10% of all cancer deaths in the UK. Nearly half of all
patients present with high-risk stage I I or stage I I I disease, and this group has an overall 5-
year survival of 33%-75% depending on the extent of local bowel and nodal involvement. For
this reason, methods to accurately stratify patients into risk categories on the basis of
persistent disease post-surgery would be particularly useful for clinical management.
Plan of investigation
Patient recruitment and sample collection
Patients with early-stage breast cancer to be treated by surgery and adjuvant therapy will be
enrolled into the trial. All such women are reviewed in a pre-surgery Oncology clinic, and it is
here that they will be approached, consented and enrolled in the study. At surgery, the breast
cancer sample will be taken by the research nurse to the Pathology lab, where a portion of
the tumour not required for diagnostic purposes will be fresh-frozen for subsequent DNA
extraction. Serial samples of 20ml_ plasma will be extracted and frozen at important
milestones during the patient's cancer care pathway: pre-surgery; post-surgery (adjuvant
therapy planning clinic); end of chemotherapy (for ER-negative patients) or during hormonal
therapy (ER-positive); 6-monthly during follow-up; at clinical relapse. Samples will be
collected for assessment of circulating tumour cells by immunological methods after surgery
and at the end of chemotherapy. Normal DNA will be extracted from whole blood leukocytes.
Patients with stage I I I and high-risk stage I I colorectal cancer undergoing resection of the
primary tumour with curative intent will be enrolled into the trial. All such patients are
managed by a specific multidisciplinary team, which includes surgeons, medical oncologists
and specialist nurses, for the whole of their staging/diagnostic phases, surgery, adjuvant
therapy and post-treatment follow-up. Patients will be consented and enrolled in the study at
pre-surgery review. At surgery, the colorectal cancer sample will be taken by the research
nurse to the Pathology lab, where a portion of the tumour not required for diagnostic
purposes will be fresh-frozen for subsequent DNA extraction. Serial samples of 20ml_ plasma
will be extracted and frozen at important milestones during the patient's cancer care pathway:
pre-surgery; post-surgery (adjuvant therapy planning clinic); end of chemotherapy; 6-monthly
during follow-up; at clinical relapse. Normal DNA will be extracted from whole blood
leukocytes.
Paired-end sequencing
Briefly, standard protocols as described above will be followed to generate libraries of 400-
500bp fragments for shotgun sequencing using 37bp paired-end reads. These will be used
for massively parallel sequencing in order to generate paired reads from 60 million fragments,
which is sufficient in our experience to identify >50% of somatic genomic rearrangements
present in the sample. Using established algorithms, we will prioritise rearrangements for
confirmatory PCR and capillary sequencing of the breakpoint - this step includes PCR across
the normal DNA sample from the patient to prove the rearrangement is somatically acquired .
Quantification of tumour-specific rearrangements in plasma DNA
Initially, 4 somatically acquired rearrangements per tumour will be taken forward for assay
design. These will be chosen on the basis of:
• Presence in majority of tumour cells - this is best estimated by taking
rearrangements demarcating integral changes in copy number (allowing for normal
cell contamination).
• Unique DNA present at the breakpoint - the absence of repeats in the PCR
amplicons will improve specificity of the assay.
• Involvement of cancer genes, where possible - for example, deletions of CDKN2A or
the first rearrangement in ERBB2 amplification are more likely to be present in all
cells, including those that ultimately relapse.
Assays will be initially based on a first round of 20 cycles of PCR with primers designed to
amplify a product no greater than 200bp (due to the small size of circulating tumour DNA
fragments), followed by a second nested round of real-time PCR with a Taqman probe.
Dilution series of tumour DNA and control amplicons will be used to estimate the relative
fraction of plasma DNA that derives from tumour cells, as well as the total amount (see figure
for examples). We have found this approach to give accurate, reproducible, linear
quantification.
DNA will be extracted in batches from frozen plasma samples using established protocols
and analysed in batches with the quantitative standards described above. Amplification of
normal control regions from the genome will be used to estimate the total amount of naked
DNA that is present in the plasma. It is likely that we will express the quantification of tumour
DNA as a fraction of total naked plasma DNA, although it may be possible to quantify
circulating tumour DNA as an absolute concentration.
Correlation with clinical outcome and power calculations
Statistical analysis will focus on three questions: Prognostic significance of individual
measurements at milestone time-points (presentation, post-surgery, at completion of
therapy); ability to quantify drug-induced cell kill by evaluation of transient increases and
subsequent fall in tumour-specific plasma DNA; and feasibility of predicting impending
relapse through identification of rising levels before clinical complications develop. Power
calculations show that for comparison of two groups of 25 patients stratified on ctDNA
estimation, a hazard ratio of 2.2 could be detected with 80% power (on the basis of a 60
month study with median disease-free survival of 30 months in the poorer prognosis group).
Additional prognostic analyses will be possible with the data set, such as correlation of
markers of overall genomic instability with outcome and association of particular patterns of
genomic rearrangement with survival.

Claims
1 A method suitable for monitoring a disease, the method comprising;
a identifying a somatically acquired genomic rearrangement associated with a disease
state in a patient, wherein the identification is carried out by genome-wide analysis of the
nucleic acid of that patient, and
b monitoring the changes in levels of nucleic acid containing the genomic
rearrangement, and/or quantifying the levels of nucleic acid containing the genomic
rearrangement, as a marker for the progression or severity of a disease in that patient.
2 A method according to claim 1 wherein the disease is a cancer.
3 A method according to claim 2 wherein the disease is a solid tumour.
4 A method according to any preceding claim wherein the nucleic acid analysis in (a) is
carried out on biopsied or surgically resected tumour tissue.
5 A method according to any preceding claim wherein the genome-wide analysis is
carried out by sequencing DNA.
6 A method according to any preceding claim wherein the genome wide analysis is a
massively parallel sequencing method.
7 A method according to claim 5 or claim 6 wherein the sequencing is carried out using
paired end sequencing.
8 A method according to any preceding claim wherein the monitoring step in (b) is a
PCR assay.
9 A method according to any preceding claim wherein the monitoring step in (b) is a
carried out on nucleic acid from a body fluid .
10 A method according to claim 1 additionally comprising the step of treating the patient
where necessary, or stopping treatment of the patient where necessary, as indicated by the
level or change in level of nucleic acid containing the genomic rearrangement.
11 A method according to claim 1 additionally comprising the step of determining a
treatment regimen for treating a patient based upon the level or change in levels of nucleic
acid containing the genomic rearrangement.
12 Use of a patient specific genomic rearrangement as a biomarker for progression of
disease in that patient.
13 Use of a monitoring process according to claim 1 in the assessment of the efficacy of
a drug or other therapeutic treatment.
14 Use of a monitoring process according to claim 13 in the assessment of the efficacy
of surgical resection of cancer tissue.
15 A method for monitoring a disease, the method comprising monitoring the changes in
levels of nucleic acid containing a genomic rearrangement, and/or quantifying the levels of
nucleic acid containing a genomic rearrangement, wherein the genomic rearrangement is a
somatically acquired mutation associated with a disease progression or severity in a patient,
and wherein the identification of the rearrangement has been carried out by genome-wide
analysis of the nucleic acid of that patient.
16 A method according to claim 1 or 15 wherein monitoring changes in levels of nucleic
acid containing the genomic rearrangement, and/or quantifying the levels of nucleic acid
containing the genomic rearrangement, are carried out on a sample from a patient in
remission.
17 A method or use according to any preceding claim wherein multiple somatically
acquired genomic rearrangements are monitored .
18 A method of medical treatment, the method comprising treating with an appropriate
treatment, or changing treatment of, or stopping treatment of, a patient in whom a change in
level of nucleic acid containing a genomic rearrangement identified according to claim 1 or 15
has been identified.