FIELD OF THE INVENTION
The invention is in the field of testing for, or aiding the detection of, surface
abnormality in the oesophagus.
BACKGROUND
Oesophageal cancer (OAC) is currently the eighth most common cancer type worldwide
and its incidence has risen almost 5-fold over the past three decades.
Barrett's oesophagus is the first step in the pathway towards OAC and meta-analyses
have demonstrated that Barrett's oesophagus confers a 0.12-0.5% increased risk of
progression to adenocarcinoma per year. Barrett's oesophagus occurs when the normal
oesophageal cells are replaced by glandular cells and this, with time, can progress to
low-grade dysplasia (LGD), high-grade dysplasia (HGD) and then finally to
adenocarcinoma.
Early diagnosis of OAC and/ or its pre-malignant precursor Barrett's oesophagus can
improve patient management and prognosis of OAC. In one known approach, the
Cytosponge™ cell collection device has been developed, for example as published in
WO2011/058316.
In addition, a test using TFF3 as a molecular marker has been developed by Fitzgerald
et a l as a clinical screening tool to detect Barrett's oesophagus, for example as
published in US20 120009597.
The first study using the Cytosponge™ (BESTl)(Kadri et al., 2010), demonstrated that
the Cytosponge™ test is a feasible method of diagnosing Barrett's oesophagus in the
primary care setting.
In the present system, symptomatic patients are sent for endoscopy. Endoscopy is an
invasive procedure requiring highly trained clinicians. It is also an uncomfortable
procedure for the patient, and can require sedation. When endoscopy is accompanied
by biopsy, there is also a degree of risk to the patient undergoing the procedure. In the
clinical setting, this is currently the only way of detecting Barrett's oesophagus, and/ or
Barrett's associated dysplasia or cancer.
Rugge et al (2010 Human Pathology vol 4 1 pages 1380-1386) disclose aurora kinase A
(AURKA) in Barrett's carcinogenesis. It is noted that TP53 mutations are recognised as
markers of an increased risk of Barrett's adenocarcinoma. Esophageal biopsy samples
were obtained from long segments of Barrett's oesophagus. 9 of 10 Barrett's
adenocarcinomas showed AURKA immunostaining. AURKA expression via mRNA
analysis and microarray studies was examined. The authors concluded by attributing a
significant role to AURKA overexpression in the progression of Barrett's mucosa to
cancer. The authors concluded that further attempts were needed in larger and
prospective studies to validate AURKAIHC expression as a potential prognostic marker
in Barrett's mucosa patients.
Liu et al (2008 World Journal of Gastroenterology vol 14 pages 7199-7207) disclose a
tissue array for Tp53, C-myc, CCND1 gene over-expression in different tumours. Seven
different tumour types were examined. Analysis was nucleic acid based. Samples used
were of known tumours. No detection method is taught. Samples were formalin fixed.
Agnese et al (2007 European Society for Medical Oncology vol 8 Suppl 6 vill0-vill5)
disclose Aurora-A overexpression as an early marker of reflux-related columnar
mucosa and Barrett's oesophagus. The authors could not find any statistically
significant quantitative differences in AURKA mRNA expression between Barrett's
mucosa (columnar lined oesophagus/ CLO) and Barrett's oesophagus (BO) with or
without dysplasia and p53 positive immunostaining.
Certain molecular markers have been studied in connection with Barrett's oesophagus.
These markers have been studied in a purely research setting. These studies have been
carried out on in vitro tissue samples. These markers have been studied singly.
Currently, no such molecular markers are used in any clinical test for Barrett's
associated abnormalities.
There is a need in the art for improved detection of Barrett's associated abnormalities.
The prior art tests are expensive and labour-intensive, invasive and involve risks to the
subject undergoing the test.
The present invention seeks to overcome problems associated with the prior art.
SUMMARY
Certain molecular markers have been shown to be associated with Barrett's associated
abnormalities. These markers have been studied on tissue biopsies. Using a molecular
marker on a tissue biopsy offers little practical advantage over the current clinical gold
standard of morphological examination of the biopsy. This is because studying the
markers in this manner still requires the biopsy to be collected, thereby still involving
each of the drawbacks associated with that invasive procedure in the prior art. More
importantly, the single markers which have been studied in the research setting have
shown inadequate sensitivity and/ or inadequate specificity to be regarded as robust
markers contributing towards detection or diagnosis.
The present inventors studied a large range of candidate markers. They also studied
these markers in different combinations. The present inventors have arrived at a small
and defined panel of markers which, when tested in combination, yield clinically useful
sensitivity and specificity scores. In addition, the inventors have studied the
performance of these markers in surface sampled cells. For example, these
combinations of markers can be employed in the analysis of cells collected from a
surface sampling of the oesophagus, such as is obtained using cell collection devices, for
example, a Cytosponge™.
The methods taught by the inventors involve novel combinations of markers which
have not previously been used in clinical tests. In addition, the inventors demonstrate
that these markers have application and produce reliable results when used on cells
obtained from surface sampling of the oesophagus. Together, these various features of
the methods of the invention provide advantages of robust and clinically useful risk
assessment, coupled to advantageously avoiding the need for invasive tissue collection
via biopsy. These and further advantages of the invention are described in more detail
below.
Thus, in a broad aspect the invention provides a method of aiding detection of a surface
abnormality in the oesophagus of a subject, the method comprising:
a) providing a sample of cells from said subject, wherein said sample
comprises cells collected from the surface of the subject's oesophagus;
b) assaying said cells for at least two markers selected from
(i) p53;
(ii) c-Myc;
(iii) AURKAor PLK1, preferably AURKA;
(iv) methylation of MyoD and Runx3; and
(v) atypia,
wherein detection of abnormal levels of at least two of said markers infers that
the subject has an increased likelihood of a surface abnormality in the
oesophagus.
In another aspect, the invention relates to a method of aiding detection of a surface
abnormality in the oesophagus of a subject, wherein said surface abnormality is
selected from the group consisting of low-grade dysplasia (LGD), high-grade dysplasia
(HGD), asymptomatic oesophageal adenocarcinoma (OAC) and intra-mucosal cancer
(IMC), the method comprising:
a) providing a sample of cells from said subject, wherein said sample
comprises cells collected from the surface of the subject's oesophagus;
b) assaying said cells for at least two markers selected from
(i) p53;
(ii) c-Myc;
(iii) AURKAor PLK1, preferably AURKA; and
(iv) methylation of MyoD and Runx3;
wherein detection of abnormal levels of at least two of said markers infers that the
subject has an increased likelihood of a surface abnormality in the oesophagus.
More suitably in one aspect the invention provides a method of aiding detection of a
surface abnormality in the oesophagus of a subject, the method comprising:
a) providing a sample of cells from said subject, wherein said sample
comprises cells collected from the surface of the subject's oesophagus;
b) assaying said cells for at least two markers selected from
(i) p53;
(ii) c-Myc;
(iii) AURKAor PLK1, preferably AURKA; and
(iv) methylation of MyoD and Runx3;
wherein detection of abnormal levels of at least two of said markers infers that
the subject has an increased likelihood of a surface abnormality in the
oesophagus.
The markers described herein are provided with guidance as to an absolute scoring for
each marker. This has the advantage of incorporating the reference
standard/ comparison phase into an already analysed scoring system. However, if
desired, the invention can instead be worked by comparison to reference standards eg.
from healthy (having no oesophageal abnormalities) subject(s). Thus, in one aspect the
invention provides a method of aiding detection of a surface abnormality in the
oesophagus of a subject, the method comprising:
a) providing a sample of cells from said subject, wherein said sample
comprises cells collected from the surface of the subject's oesophagus;
b) assaying said cells for at least two markers selected from
(i) p53;
(ii) c-Myc;
(iii) AURKA; and
(iv) methylation of MyoD and Runx3;
wherein detection of abnormal levels of at least two of said markers compared
to a reference standard infers that the subject has an increased likelihood of a
surface abnormality in the oesophagus.
Optionally step (b) comprises
(1) contacting said cells with reagents for detection of at least a first
molecular marker selected from:
(i) p53;
(ii) c-Myc;
(iii) AURKA; and
(iv) methylation of MyoD and Runx3, and
(2) contacting said cells with reagents for detection of at least a second
molecular marker selected from (i) to (iv) and/ or assaying said cells for atypia.
More suitably step (b) comprises
(1) contacting said cells with reagents for detection of at least a first
molecular marker selected from:
(i) p53;
(ii) c-Myc;
(iii) AURKA; and
(iv) methylation of MyoD and Runx3, and
(2) contacting said cells with reagents for detection of at least a second molecular
marker selected from (i) to (iv).
Optionally said surface abnormality is selected from the group consisting of low-grade
dysplasia (LGD), high-grade dysplasia (HGD), asymptomatic oesophageal
adenocarcinoma (OAC) and intra-mucosal cancer (IMC). These all share the property
of being 'glandular' ('columnar'). These all share the property of being Barrett's'.
These are all dysplasia. None of these are squamous.
Suitably the invention is not concerned with squamous cell dysplasia.
Suitably the invention is not concerned with squamous cell cancer.
Suitably the surface abnormality is not a squamous cell abnormality.
Optionally said surface abnormality is selected from the group consisting of low-grade
dysplasia (LGD), high-grade dysplasia (HGD), and intra-mucosal cancer (IMC).
Optionally said surface abnormality is selected from the group consisting of low-grade
dysplasia (LGD) and high-grade dysplasia (HGD).
Optionally said surface abnormality is selected from the group consisting of
asymptomatic oesophageal adenocarcinoma (OAC) and intra-mucosal cancer (IMC).
Optionally said surface abnormality is low-grade dysplasia (LGD).
Optionally said surface abnormality is high-grade dysplasia (HGD).
Optionally said surface abnormality is asymptomatic oesophageal adenocarcinoma
(OAC).
Optionally said surface abnormality is intra-mucosal cancer (IMC).
Optionally abnormal levels of at least three of said markers are assayed.
Optionally abnormal levels of at least four of said markers are assayed.
Optionally abnormal levels of each of said markers are assayed.
Optionally said cells are collected by unbiased sampling of the surface of the
oesophagus.
Optionally said cells are collected using a capsule sponge.
Optionally the cells are prepared prior to being contacted with the reagents for
detection of the molecular markers by the steps of (i) pelleting the cells by centrifuge,
(ii) re-suspending the cells in plasma, and (iii) adding thrombin and incubating until a
clot is formed. Optionally preparation further comprises the step of incubating said
clot in formalin, processing into a paraffin block, and slicing into sections suitable for
microscopic examination.
Optionally p53 is assessed by immunohistochemistry.
Optionally p53 is assessed at the nucleic acid level. Optionally p53 mutation status is
assessed (e.g. detected). Optionally p53 mutations are assessed (e.g. detected) by
sequencing. Suitably when p53 is detected at the nucleic acid level, 'detection of
abnormal levels' means detection of a p53 mutation. In other words, detection of a p53
mutation is itself regarded as an abnormal p53 or abnormal level of p53. Assessing p53
at the nucleic acid level has the advantage of removing or ameliorating subjectivity
which can be present when assessing staining levels e.g. at the protein level for p53.
Suitably p53 mutation(s) anywhere within the p53 gene are detected. This is
advantageous since mutation(s) can be widespread throughout the gene. More suitably
mutations in the DNA binding domain are detected. These are the most common
mutations. Suitably the assay is capable of detecting mutations throughout the gene -
see example 10 for more detail if further guidance is needed.
Suitably a p53 mutation is detected when a p53 nonsense mutation is detected. Suitably
a p53 mutation is detected when a p53 missense mutation is detected. Suitably a p53
mutation is detected when a p53 deletion mutation is detected. Suitably a p53 mutation
is detected when a p53 INDEL variant mutation is detected.
Suitably the p53 mutation is one mentioned in Example 10.
Suitably the p53 mutation is one in the DNAbinding domain of p53.
Optionally p53 is assessed at both the nucleic acid and the protein level. This provides
the advantage that any mutations which are not detected by protein assay are caught
(E.g. p53 mutations which do not affect p53 expression/ detection), and also any nonp53
changes (e.g. mutations in genes other than p53) which affect p53 expression are
also caught (i.e. by the protein analysis).
Suitably p53 is assessed by detection of one or more p53 mutation(s).
Suitably p53 is assessed by immunohistochemistry and p53 is also assessed by
detection of one or more p53 mutation(s).
Optionally cMyc is assessed by immunohistochemistry.
Optionally AURKAis assessed by immunohistochemistry.
It should be noted that AURKA is a preferred marker of the invention. However it will
be appreciated that marker PLK1 also has a good sensitivity (91%) and a good
specificity (88%). This biomarker was excluded in favour of AURKA as AURKA gave
better sensitivity (93%) and specificity (94%) data (see examples). However, the
inventors teach that AURKA or PLK1 overexpression detect essentially the same cases.
Therefore in embodiments of the invention PLK1 may be assayed instead of (or in
addition to) AURKA. Thus suitably AURKAor PLK1 is assayed, preferably AURKA.
Optionally methylation of MyoD/Runx3 is assessed by MethyLight analysis.
Optionally atypia is assessed by scoring the cells for their morphology according to the
Vienna Scale. Suitably the Vienna scale is as described in Schlemper et al 2007 Gut
2000;47:251-255.
In another aspect, the invention relates to a method as described above wherein step
(b) of said method is preceded by the step of assaying said cells for TFF3.
In another aspect, the invention relates to an assay for selecting a treatment regimen,
said assay comprising
a) providing a sample of cells from said subject, wherein said sample
comprises cells collected from the surface of the subject's oesophagus;
b) assaying said cells for at least two markers selected from
(i) p53;
(ii) c-Myc;
(iii) AURKA; and
(iv) methylation of MyoD and Runx3;
wherein if abnormal levels of at least two of said markers are detected, then a treatment
regimen of endoscopy and biopsy is selected.
In another aspect, the invention relates to an apparatus or system which is
(a) configured to analyse an oesophagal sample from a subject, wherein said
analysis comprises
(b) assaying said cells for at least two markers selected from
(i) p53;
(ii) c-Myc;
(iii) AURKA; and
(iv) methylation of MyoD and Runx3;
said apparatus or system comprising an output module,
wherein if abnormal levels of at least two of said markers are detected, then said output
module indicates an increased likelihood of a surface abnormality in the oesophagus for
said subject.
In another aspect, the invention relates to use for applications relating to aiding
detection of a surface abnormality in the oesophagus of a subject, of a material which
recognises, binds to or has affinity for certain polypeptides, or methylation of certain
nucleic acid sequences, wherein the polypeptides and/ or nucleic acid sequences are as
defined as above eg. p53, c-Myc, AURKA, methylation of Runx3/MyoDl. In another
aspect, the invention relates to such a use of a combination of materials, each of which
respectively recognises, binds to or has affinity for one or more of said polypeptide(s) or
nucleic acid sequences.
In another aspect, the invention relates to an assay device for use in aiding detection of
a surface abnormality in the oesophagus of a subject, which comprises a solid substrate
having a location containing a material, which recognises, binds to or has affinity for
certain polypeptides, or methylation of certain nucleic acid sequences, wherein the
polypeptides and/ or nucleic acid sequences are as defined above eg. p53, c-Myc,
AURKA, methylation of Runx3/MyoDl.
In another aspect, the invention relates to a kit comprising reagents for determining the
expression level of each of
(i) p53;
(ii) c-Myc;
(iii) AURKA;
in a biological sample, and optionally further comprising reagents for determining the
methylation of MyoD and Runx3.
In another aspect, the invention relates to a method for aiding the detection of a surface
abnormality in the oesophagus of a subject, the method comprising providing a sample
of cells from said subject, wherein said sample comprises cells collected from the
surface of the subject's oesophagus, assaying said cells for TFF3, wherein if TFF3 is
detected in cell(s) of the sample, the method as described above carried out, wherein
detection of abnormal levels of at least one marker in addition to detection of TFF3
indicates an increased likelihood of a surface abnormality in the oesophagus of said
subject.
In another aspect, the invention relates to a method for aiding the detection of a surface
abnormality in the oesophagus of a subject, the method comprising
(a) providing a sample of cells from said subject, wherein said sample comprises cells
collected from the surface of the subject's oesophagus, assaying said cells for TFF3,
wherein if TFF3 is detected in cell(s) of the sample, then the following additional steps
are performed:
(b) assaying said cells for at least two markers selected from
(i) p53;
(ii) c-Myc;
(iii) AURKA; and
(iv) methylation of MyoD and Runx3;
wherein detection of abnormal levels of at least one marker in addition to detection of
TFF3 indicates an increased likelihood of a surface abnormality in the oesophagus of
said subject. Optionally detection of abnormal levels of at least two markers in addition
to detection of TFF3, preferably least three markers in addition to detection of TFF3,
preferably least four markers in addition to detection of TFF3, preferably each of the
markers in addition to detection of TFF3, indicates an increased likelihood of a surface
abnormality in the oesophagus of said subject. Optionally said cells are collected by
unbiased sampling of the surface of the oesophagus. Optionally said cells are collected
using a capsule sponge.
In another aspect, the invention relates to a method of collecting information useful for
detecting oesophageal abnormalities comprising carrying out the steps as described
above.
In another aspect, the invention relates to a method of collecting information useful for
aiding diagnosis of oesophageal abnormalities comprising carrying out the steps as
described above.
In another aspect, the invention relates to a method of diagnosis of oesophageal
abnormalities comprising carrying out the steps as described above.
In another aspect, the invention relates to a method of aiding diagnosis of oesophageal
abnormalities comprising carrying out the steps as described above.
In another aspect, the invention relates to a method of assessing the risk of oesophageal
abnormalities comprising carrying out the steps as described above.
In another aspect, the invention relates to a method of assessing the risk of an
oesophageal abnormality comprising carrying out the steps as described above.
Optionally said abnormality is dysplasia. Optionally said abnormality is LGD, HGD,
IMC or asymptomatic OAC.
In another aspect, the invention relates to a method for aiding the detection of a surface
abnormality in the oesophagus of a subject, wherein said surface abnormality is
oesophageal adenocarcinoma (OAC), the method comprising providing a sample of
cells from said subject, wherein said sample comprises cells collected from the surface
of the subject's oesophagus, assaying said cells for SMAD4, wherein if SMAD4 is
detected in cell(s) of the sample an increased likelihood of oesophageal
adenocarcinoma (OAC) in the oesophagus of said subject is indicated.
DETAILED DESCRIPTION OF THE INVENTION
The invention finds particular application in the assessment of the risk of a subject
having dysplasia. Currently the assessment of dysplasia is only performed on biopsies
collected from the subject. According to the present invention the subject can be
assessed for their risk of having dysplasia (such as one or more of LGD, HGD, IMC;
optionally also including asymptomatic OAC) by the methods described herein. These
methods advantageously avoid biopsy. The methods of the invention suitably expressly
exclude biopsy. The methods of the invention advantageously require only surface
sampling of the oesophagus (or an in vitro sample from the surface of the oesophagus),
thereby avoiding biopsy and/ or endoscopy.
Thus a key part of the invention is the use of the panel of markers to assess the risk of
the subject having dysplasia such as one or more of LGD, HGD, or IMC.
OACis more typically regarded as an invasive form of disease; typically patients with
OAC already display symptoms; typically the methods of the invention are used for
screening or surveillance applications and for risk assessment applications rather than
for express diagnosis of (e.g.) OAC. Invasive OACis typically diagnosed using a
different algorithm which is not part of this invention. However, asymptomatic OAC
(or more precisely the elevated risk of asymptomatic OAC) can be detected by the
methods of the present invention in the same manner as LGD/ HGD/ IMC (or more
precisely the elevated risk of LGD/ HGD/ IMC). This has been carried out by the
inventors. The invention was applied in the manner described herein. The result of
that application of the method was an indication of higher risk of
abnormality/ dysplasia in that subject. The subject was recommended to undergo
endoscopy/ biopsy as a result of the finding of higher risk according to the present
invention. The endoscopy/ biopsy revealed asymptomatic OAC. The patient was then
referred for appropriate treatment. Therefore the invention can be applied to the
assessment of risk of abnormality/ dysplasia which can include asymptomatic OAC, but
the invention does not purport to be a diagnostic tool giving a definite diagnosis of
OAC.
Subject/Patient Groups
Suitably the methods of the invention are applied to any subject. Suitably the methods
of the invention are applied to any subject suspected of having Barrett's oesophagus.
These applications might be useful in screening the population at large.
More suitably the methods/ panel of the invention finds application in subjects or
patients who are not known to have carcinoma but may be monitored or followed-up
for Barrett's oesophagus.
More suitably the methods of the invention are applied to any subject having Barrett's
oesophagus.
It is aiding the assessment of the risk of progression or the risk of having
LGD/HGD/IMC in subjects which already have Barrett's oesophagus which is a key
benefit of the invention.
The panel of the invention is not intended for detection of Barrett's oesophagus, but is
intended for assessment of the risk of having dysplasia. Assessment of having Barrett's
oesophagus is typically carried out using the established TFF3 marker of Barrett's
oesophagus, or may be carried out by any suitable method for diagnosis of Barrett's
oesophagus.
Akey marker of Barrett's oesophagus is the TFF3 marker (ie TFF3 positive on the
surface sampled cells eg from a capsule sponge such as a Cytosponge™. Such subjects
may turn out to have no dysplasia, low grade dysplasia, high grade dysplasia, or be
indefinite for dysplasia. It is also possible that the patient could have an undiagnosed
superficial intramucosal carcinoma. However the main benefit of the invention is in
assessing risk of having dysplasia from a start point of already having Barrett's
oesophagus. Of course the panel/method of the invention can be applied as a general
screening tool to asymptomatic subjects, but this might not be economic (even though
it would of course be very effective). Thus for economic and practical reasons the
invention finds best application in screening those subjects already at risk of dysplasia,
ie. those patients already having Barrett's oesophagus.
Suitably the subject has Barrett's oesophagus.
Suitably the subject tests positive for TFF3 in surface sampled oesophagus cells.
In one embodiment the test (panel) of the invention may be preceded by testing for
TFF3. This serves as a useful internal control. If the subject is known to have Barrett's
oesophagus, then their surface sampled cells should test positive for TFF3. Therefore if
a surface sample of the oesophagus of a subject who is known to have Barrett's
oesophagus tests negative for TFF3, this would indicate that the sample is inadequate
(eg. insufficient cells, or lack of columnar cells, or some other issue). The
recommendation then would be to resample the surface of the subject's oesophagus
and retest for TFF3, and only proceed to test using the panel of the invention once a
positive result for TFF3 is observed, indicating a reliable/robust sample from a patient
with Barrett's oesophagus.
The inventors have, among other things, designed BEST2, a multicentre, prospective
case and control study aiming to recruit 1,000 patients which is carried out to test the
performance characteristics of the Cytosponge™ for diagnosing Barrett's oesophagus
compared with endoscopy. Additionally, within BEST2, a panel of risk stratification
biomarkers are evaluated on the Cytosponge™ to determine their ability to risk stratify
patients according to the endoscopic grade of dysplasia. The panel of risk stratification
biomarkers consists of four different biomarkers, namely p53 protein levels, c-MYC
protein levels, Aurora kinase A (AURKA) protein levels and methylation of the
promoter regions of the Runt-related transcription factor 3 (RUNX3) and myogenic
differentiation 1 (MYOD1) genes. Optionally the panel may further comprise a fifth
marker, atypia.
Sample and Sample Collection
Suitably the sample comprises cells from the subject of interest. Suitably the sample
comprises oesophageal cells from the subject of interest. Suitably the sample is nonendoscopic
ie. suitably the sample is obtained without the use of an endoscope.
Endoscopic sampling is an invasive technique. Furthermore, endoscopic sampling is a
targeted technique where biopsies are taken at intervals along the oesophagus, or
where lesions are visually identified by the operator and specifically targeted for biopsy.
Suitably the invention does not involve endoscopic samples such as endoscopic
biopsies.
Akey principle of the invention is to provide a test which is specific for oesophageal
abnormalities. The test is specific for in the sense of not delivering problematic levels of
false positives from cells of unrelated tissues such as normal squamous oesophagus, or
gastric cardia (stomach). Thus, by providing a test with these specific characteristics,
the invention advantageously provides a test targeted to detection of abnormal
oesophagus cells. In this way, the invention advantageously avoids the need for
targeted sample collection. Thus, the invention advantageously involves samples
obtained by non-targeted sample collection such as sampling the entire surface of the
oesophagus rather than only targeting areas of suspected lesions (Barrett's).
Thus, suitably the sample does not comprise an endoscopic biopsy.
Suitably the sample may comprise oesophageal brushings or surface cells. Oesophageal
brushings may be obtained using an endoscope or by other means; suitably when the
sample comprises oesophagal brushings they are obtained by non-endoscopic means.
Suitably the sample comprises cells from the surface of a subject's upper intestinal
tract.
Suitably the sample consists of cells from the surface of a subject's upper intestinal
tract.
Suitably the sample may comprise cells sampled from the entire oesophageal lumen.
Suitably the sample may comprise both oesophageal and non-oesophageal cells.
Suitably the sample may comprise oesophageal cells together with gastric cardia cells.
Suitably the sample may consist of oesophageal cells.
Suitably the sample comprises cells from the surface of a subject's oesophagus.
Suitably the sample consists of cells from the surface of a subject's oesophagus.
Most suitably, the sample may comprise cells collected using a capsule sponge type
sampling technique.
Especially suitable sampling techniques are described in the examples section.
Examples of suitable samples include oesophageal brushings (whether endoscopically
or non-endoscopically obtained), samples obtained via balloon cytology, samples
obtained via capsule sponge sampling. Most suitably, a sample comprises cells obtained
via capsule sponge sampling.
The panel of markers are relevant to luminal surface cells. This means that the sample
to be analysed need only be collected from the surface of the oesophageal lumen. This
advantageously avoids the need for a biopsy such as an endoscopic biopsy. Moreover,
this advantageously avoids the need to preserve tissue architecture in the sample being
analysed.
Afurther advantage of the markers of the invention is that they have been selected to
avoid false positives arising from cells collected from the gastric mucosa (e.g. gastric
cardia/ stomach). This has a specific advantage that if cells of the gastric mucosa are
included in the sample, then the panel will still able to function as a mode of detection
of oesophageal abnormalities. This is because the markers are not found in gastric
mucosa cells, and therefore no false positives occur even when the sample comprises
cells of the gastric mucosa.
Thus it can be appreciated that the choice of markers in the panel by the inventors
provides a degree of specificity which has not yet been provided in any prior art
approach to screening for oesophageal abnormalities. The present inventors were the
first to actively seek, and to successfully provide, a panel capable of such focused
discrimination.
Anon-endoscopic capsule sponge device which has been used in a previous clinical
study (for example Ref no: CI/ 2007/ 0053 in the UK) may be used for sample
collection. Apilot study demonstrated that this device (the 'Cytosponge™') is
acceptable to patients and could be used in primary care. The device consists of a
polyurethane sponge, contained within a gelatin capsule, which is attached to a string.
The capsule is swallowed and dissolves within the stomach after 3-5 minutes.
Suitably the cytological specimen collected is processed to a pellet which can then be
embedded in paraffin thus preserving the tissue architecture. This can then undergo
histological assessment and in addition, multiple molecular and/ or morphological
markers may be used on a single sample. Thus, this mode of sample collection is
particularly suitable for use in the present invention.
The cells are suitably sampled from the surface of the oesophagus using a swallowable
abrasive material, which material is retrieved from the patient and from which the cells
are subsequently separated for analysis to determine the presence of the markers.
Preferably substantially the entire surface of the oesophagus is sampled, preferably the
entire surface.
By abrasive is meant that the material is capable of removing cells from the internal
surface of the oesophagus. Clearly, since this is meant for use in a subject's oesophagus,
abrasive" must be interpreted in the light of the application. In the context of the
present invention the term abrasive" has the meaning given above, which can be
tested by passing the material through the oesophagus in an appropriate
amount/ configuration and examining it to determine whether cells have been removed
from the oesophagus.
The material used in the collection device must be sufficiently abrasive to sample any
dysplastic cells present in the oesophagus. Preferably the material is sufficiently
abrasive to sample any Barrett's or dysplastic or adenocarcinoma cells present. In a
most preferred embodiment, preferably the material is sufficiently abrasive to be
capable of sampling the whole oesophagus ie. so that some squamous cells are collected
together with any Barrett's and/ or columnar and/ or adenocarcinoma cells which may
be present. This is advantageous because squamous cells are more difficult to remove
than dysplastic cells and so their sampling provides a control to the operator such that
if normal squamous cells are removed by the material then the chances of having not
sampled the cells of interest such as Barrett's or dysplastic cells (if present), which are
easier to remove than normal squamous cells, is correspondingly small.
Preferably the swallowable abrasive material is expandable. In this embodiment,
preferably the abrasive material is of a smaller size when swallowed than when
withdrawn. An expandable material may be simply a resilient material compressed
such that when released from compression it will expand again back to a size
approximating its uncompressed size. Alternatively it may be a material which expands
e.g. upon taking up aqueous fluid to a final size exceeding its original size.
In other words, preferably the material of the device expands, swells, inflates or
otherwise increases in size between swallowing and withdrawal. Preferably the device is
auto-expandable ie. does not require further intervention between swallowing and
expansion. Preferably the device is not inflatable. Preferably the device expands by
unfolding, unfurling, uncoiling or otherwise growing in size following removal of
restraint after swallowing. Preferably the material of the device is compressible and
reverts a size approximating its uncompressed size following swallowing. Preferably the
device is constructed from a compressed material which is releasably restrained in a
compressed state. Preferably the material is released from restraint after swallowing,
allowing expansion of the device/ material before withdrawal.
Preferably the device comprises compressible material which is compressed into
capsule form. Preferably the compressible material is in the form of sponge material.
Preferably the compressed sponge is at least partially surrounded by a soluble and/ or
digestible coat such as a capsule coat. Preferably the sponge is indigestible. Preferably
the capsule coat is at least partially formed from gelatin. Preferably the capsule coat is
fully formed from gelatin.
In one embodiment it may be desirable to make the whole device out of digestible
material to increase safety in case of a device becoming lost in the subject. Naturally the
abrasive material would need to be digested at a slower rate than the capsule and the
cord would need to be similarly slowly digested. Preferably the abrasive material is
non-digestible. Preferably the cord is non-digestible.
Preferably the abrasive material comprises polyurethane, preferably polyurethane
sponge.
Suitably said abrasive material is compressible. Suitably said abrasive material
comprises reticulated polyurethane.
Suitably the material has a uniform shape.
Suitably the material has a uniform diameter.
Suitably the uncompressed shape is round such as spherical.
Suitably the uncompressed diameter is 3cm.
Suitably said cord is attached to said abrasive material via a loop of cord arranged
below the surface of the abrasive material, said loop being closed by a hitch knot.
Suitably said abrasive material is compressed and wherein said abrasive material is
retained in a compressed state by a soluble capsule.
Suitably said soluble capsule comprises a gelatine capsule.
Suitably said capsule is capable of dissolution and the compressible abrasive material is
capable of reverting to its uncompressed size within 5 minutes upon immersion in
water at 30 degrees Celsius.
Preferably the device is a capsule sponge. As will be apparent from the specification, a
capsule sponge is a device comprising compressible sponge as the abrasive material,
which sponge is compressed into a capsule shape, which capsule shaped compressed
sponge is preferably reversibly restrained in its compressed state by at least a partial
coat of soluble and/ or digestible material such as gelatine. Preferably the device is a
capsule sponge as described in WO2011/058316.
Preferably the sample does not comprise endoscopically collected material. Preferably
the sample does not comprise endoscopic biopsy. Preferably the sample does not
comprise endoscopic brushings.
It is a feature of the invention that the sampling is not directed e.g. visually directed to
any particular part of the oesophagus but rather the sponge is scraped along the entire
surface of the oesophagus and obtains a heterogeneous sample of cells from the tract.
It is a further advantage of the invention that a greater proportion of the surface of the
oesophagus is sampled than is achieved by prior art techniques such as endoscopic
biopsy (which samples approximately 1% of the surface) or endoscopic brushing.
Preferably at least 10% of the oesophageal surface is sampled, preferably at least 20%,
preferably at least 30%, preferably at least 40%, preferably at least 50%, preferably at
least 60%, preferably at least 70%, preferably at least 80%, preferably at least 90%. In a
most preferred embodiment, preferably substantially the entire oesophagus is sampled,
preferably the whole inner lumen of the oesophagus is sampled. This applies equally to
the in vitro sample e.g. when the method of the invention does not include collection of
the sample.
Suitably the sample is an in vitro sample.
Suitably the sample is an extracorporeal sample.
Suitably sampling the cellular surface of the upper intestinal tract such as the
oesophagus comprises the steps of
(i) introducing a swallowable device comprising abrasive material capable of
collecting cells from the surface of the oesophagus into the subject,
(ii) retrieving said device by withdrawal through the oesophagus, and
(iii) collecting the cells from the device.
Preferably step (i) comprises introducing a swallowable device comprising abrasive
material capable of collecting cells from the surface of the oesophagus into the subject's
stomach.
Suitably the sample is from a white Caucasian human subject.
Suitably the sample is from a subject with a history of reflux.
Suitably the sample is from a male subject.
Suitably the sample is from an obese subject.
Methods of the Invention
In one embodiment suitably the method is an in vitro method. In one embodiment
suitably the method is an extracorporeal method. In one embodiment suitably the
actual sampling of the cells is not part of the method of the invention. Suitably the
method does not involve collection of the cells.
Suitably the sample is a sample previously collected. Suitably the method does not
require the presence of the subject whose cells are being assayed. Suitably the sample
is an in vitro sample. Suitably the method does not involve the actual medical decision,
stricto sensu; such a decision stricto sensu would typically be taken by the physician.
Suitably the method of the invention is conducted in vitro. Suitably the method of the
invention is conducted extracorporeal^.
Markers Used in the Invention
Marker Abbreviation Accession number Comments
/sequence
Trefoil factor 3 TFF3 NM_003226.2 protein
p53 tumour p53 NM_000546 protein
suppressor
protein
p53 tumour p53 NM_000546 nucleic acid
suppressor (most suitably
protein version
NM_000546.5 -
shown in full below)
c-Myc oncogene c-Myc NM_002467 protein
Aurora kinase A AURKA NM_ 198434 protein
most suitably the
AURKA accession
number/sequence is
NP_003591.2, which
corresponds to the
AURKA associated
with the exemplary
antibody used (see
examples)
serine/ threonine- PLK1 NP_005021.2 protein
protein kinase
PLK1
myogenic MyoDl NM_002478 methylation
differentiation 1 (see examples for of nucleic
primer sequences acid
defining target)
Runt-related Runx3 NM_001031680 methylation
transcription (see examples for of nucleic
factor 3 primer sequences acid
defining target)
The Genbank accession numbers are provided with reference to the database as of the
filing date of this application ie. 2 1Feb 20 13. In case any further assistance is needed,
preferably the accession numbers provided should be taken to refer to Genbank release
number 194.0 of 15 Feb 20 13.
By way of illustration, the exemplary p53 sequence is provided below, as retrieved from
GenBank:
Homo sapiens tumor protein p53 (TP53), transcript variant 1, mRNA
NCBI Reference Sequence: NM_000546.5
ACCESSION NM_000546
VERSION NM_000546.5
ORIGIN
1 gatgggattg gggttttccc ctcccatgtg ctcaagactg gcgctaaaag ttttgagctt
61 ctcaaaagtc tagagccacc gtccagggag caggtagctg ctgggctccg gggacacttt
121 gcgttcgggc tgggagcgtg ctttccacga cggtgacacg cttccctgga ttggcagcca
181 gactgccttc cgggtcactg ccatggagga gccgcagtca gatcctagcg tcgagccccc
241 tctgagtcag gaaacatttt cagacctatg gaaactactt cctgaaaaca acgttctgtc
301 ccccttgccg tcccaagcaa tggatgattt gatgctgtcc ccggacgata ttgaacaatg
361 gttcactgaa gacccaggtc cagatgaagc tcccagaatg ccagaggctg ctccccccgt
421 ggcccctgca ccagcagctc ctacaccggc ggcccctgca ccagccccct cctggcccct
481 gtcatcttct gtcccttccc agaaaaccta ccagggcagc tacggtttcc gtctgggctt
541 cttgcattct gggacagcca agtctgtgac ttgcacgtac tcccctgccc tcaacaagat
601 gttttgccaa ctggccaaga cctgccctgt gcagctgtgg gttgattcca cacccccgcc
661 cggcacccgc gtccgcgcca tggccatcta caagcagtca cagcacatga cggaggttgt
721 gaggcgctgc ccccaccatg agcgctgctc agatagcgat ggtctggccc ctcctcagca
781 tcttatccga gtggaaggaa atttgcgtgt ggagtatttg gatgacagaa acacttttcg
841 acatagtgtg gtggtgccct atgagccgcc tgaggttggc tctgactgta ccaccatcca
901 ctacaactac atgtgtaaca gttcctgcat gggcggcatg aaccggaggc ccatcctcac
961 catcatcaca ctggaagact ccagtggtaa tctactggga cggaacagct ttgaggtgcg
1021 tgtttgtgcc tgtcctggga gagaccggcg cacagaggaa gagaatctcc gcaagaaagg
1081 ggagcctcac cacgagctgc ccccagggag cactaagcga gcactgccca acaacaccag
1141 ctcctctccc cagccaaaga agaaaccact ggatggagaa tatttcaccc ttcagatccg
1201 tgggcgtgag cgcttcgaga tgttccgaga gctgaatgag gccttggaac tcaaggatgc
1261 ccaggctggg aaggagccag gggggagcag ggctcactcc agccacctga agtccaaaaa
1321 gggtcagtct acctcccgcc ataaaaaact catgttcaag acagaagggc ctgactcaga
1381 ctgacattct ccacttcttg ttccccactg acagcctccc acccccatct ctccctcccc
1441 tgccattttg ggttttgggt ctttgaaccc ttgcttgcaa taggtgtgcg tcagaagcac
1501 ccaggacttc catttgcttt gtcccggggc tccactgaac aagttggcct gcactggtgt
1561 tttgttgtgg ggaggaggat ggggagtagg acataccagc ttagatttta aggtttttac
1621 tgtgagggat gtttgggaga tgtaagaaat gttcttgcag ttaagggtta gtttacaatc
1681 agccacattc taggtagggg cccacttcac cgtactaacc agggaagctg tccctcactg
1741 ttgaattttc tctaacttca aggcccatat ctgtgaaatg ctggcatttg cacctacctc
1801 acagagtgca ttgtgagggt taatgaaata atgtacatct ggccttgaaa ccacctttta
1861 ttacatgggg tctagaactt gacccccttg agggtgcttg ttccctctcc ctgttggtcg
1921 gtgggttggt agtttctaca gttgggcagc tggttaggta gagggagttg tcaagtctct
1981 gctggcccag ccaaaccctg tctgacaacc tcttggtgaa ccttagtacc taaaaggaaa
2041 tctcacccca tcccacaccc tggaggattt catctcttgt atatgatgat ctggatccac
2101 caagacttgt tttatgctca gggtcaattt cttttttctt tttttttttt ttttttcttt
2161 ttctttgaga ctgggtctcg ctttgttgcc caggctggag tggagtggcg tgatcttggc
2221 ttactgcagc ctttgcctcc ccggctcgag cagtcctgcc tcagcctccg gagtagctgg
2281 gaccacaggt tcatgccacc atggccagcc aacttttgca tgttttgtag agatggggtc
2341 tcacagtgtt gcccaggctg gtctcaaact cctgggctca ggcgatccac ctgtctcagc
2401 ctcccagagt gctgggatta caattgtgag ccaccacgtc cagctggaag ggtcaacatc
2461 ttttacattc tgcaagcaca tctgcatttt caccccaccc ttcccctcct tctccctttt
2521 tatatcccat ttttatatcg atctcttatt ttacaataaa actttgctgc cacctgtgtg
2581 tctgaggggt g
Atypia
Atypia is assessed by observation.
Suitably the cells are stained before observation. Suitably the cells are stained using
haematoxylin and eosin (H&E) stain. This has the advantage of rendering the cells
easily distinguished from one another according to conventional and long established
histology.
Standard histology / cytology is used to tell the cells apart.
Scoring is carried out in accordance with the Vienna scale.
In the context of the invention, abnormal is judged according to the Vienna scale;
therefore observing one or more of those abnormal categories of cells when assaying
atypia as an optional extra marker in addition to the panel of markers of the invention
would mean that a finding of 'abnormal' was recorded for the atypia marker in that
analysis.
It is an advantage of optionally also assaying atypia in addition to the four markers of
the panel of the invention that increased sensitivity and/ or specificity may be obtained.
In case any further guidance is needed, reference is made to standard text books in this
area such as Diagnostic Cytopathology by Winifred Gray 2nd edition. In addition, or
alternatively, text books such as Gastrointestinal Pathology An Atlas and Textbook by
Cecilia M. Fenoglio-Preiser, Amy E. Noffsinger, Grant N. Stemmermann, Patrick E.
Lantz, Peter G. Isaacson Third edition may be used. These texts are specifically

CLAIMS
1. A method of aiding detection of a surface abnormality in the oesophagus of a
subject, wherein said surface abnormality is selected from the group consisting of lowgrade
dysplasia (LGD), high-grade dysplasia (HGD), asymptomatic oesophageal
adenocarcinoma (OAC) and intra-mucosal cancer (IMC), the method comprising:
a) providing a sample of cells from said subject, wherein said sample
comprises cells collected from the surface of the subject's oesophagus;
b) assaying said cells for at least two markers selected from
(i) p53;
(ii) c-Myc;
(iii) AURKAor PLK1, preferably AURKA; and
(iv) methylation of MyoD and Runx3;
wherein detection of abnormal levels of at least two of said markers infers that
the subject has an increased likelihood of a surface abnormality in the
oesophagus.
Amethod according to claim 1wherein step (b) comprises
(1) contacting said cells with reagents for detection of at least a first
molecular marker selected from:
(i) p53;
(ii) c-Myc;
(iii) AURKAor PLK1, preferably AURKA; and
(iv) methylation of MyoD and Runx3, and
(2) contacting said cells with reagents for detection of at least a second
molecular marker selected from (i) to (iv).
3. A method according to claim 1 or claim 2, wherein abnormal levels of at least
three of said markers are assayed.
4. A method according to any preceding claim, wherein abnormal levels of at least
four of said markers are assayed.
5. A method according to any preceding claim, further comprising assaying said
cells for atypia.
6. A method according to any preceding claim, wherein said cells are collected by
unbiased sampling of the surface of the oesophagus.
7. A method according to claim 6, wherein said cells are collected using a capsule
sponge.
8. A method according to any preceding claim, wherein the cells are prepared
prior to being contacted with the reagents for detection of the molecular markers by the
steps of (i) pelleting the cells by centrifuge, (ii) re-suspending the cells in plasma, and
(iii) adding thrombin and incubating until a clot is formed.
9. A method according to claim 8, further comprising the step of incubating said
clot in formalin, processing into a paraffin block, and slicing into sections suitable for
microscopic examination.
10. A method according to any of claims 1 to 9, wherein p53 is assessed by
immunohistochemistry.
11. Amethod according to any of claims 1to 9, wherein p53 is assessed by detection
of one or more p53 mutation(s).
12. A method according to any of claims 1 to 9, wherein p53 is assessed by
immunohistochemistry and wherein p53 is also assessed by detection of one or more
p53 mutation(s).
13. A method according to any preceding claim, wherein cMyc is assessed by
immunohistochemistry.
14. A method according to any preceding claim, wherein AURKA is assessed by
immunohistochemistry.
15. A method according to any preceding claim, wherein methylation of
MyoD/Runx3 is assessed by MethyLight analysis.
16. A method according to claim 6, wherein atypia is assessed by scoring the cells
for their morphology according to the Vienna Scale.
17. A method according to any preceding claim, wherein step (b) of said method is
preceded by the step of assaying said cells for TFF3.
18 . An assay for selecting a treatment regimen, said assay comprising
a) providing a sample of cells from said subject, wherein said sample
comprises cells collected from the surface of the subject's oesophagus;
b) assaying said cells for at least two markers selected from
(i) p53;
(ii) c-Myc;
(iii) AURKA; and
(iv) methylation of MyoD and Runx3;
wherein if abnormal levels of at least two of said markers are detected, then a treatment
regimen of endoscopy and biopsy is selected.
19. An apparatus or system which is
(a) configured to analyse an oesophagal sample from a subject, wherein said
analysis comprises
(b) assaying said cells for at least two markers selected from
(i) p53;
(ii) c-Myc;
(iii) AURKA; and
(iv) methylation of MyoD and Runx3;
said apparatus or system comprising an output module,
wherein if abnormal levels of at least two of said markers are detected, then said output
module indicates an increased likelihood of a surface abnormality in the oesophagus for
said subject, wherein said surface abnormality is selected from the group consisting of
low-grade dysplasia (LGD), high-grade dysplasia (HGD), asymptomatic oesophageal
adenocarcinoma (OAC) and intra-mucosal cancer (IMC).
20. Use for applications relating to aiding detection of a surface abnormality in the
oesophagus of a subject, wherein said surface abnormality is selected from the group
consisting of low-grade dysplasia (LGD), high-grade dysplasia (HGD), asymptomatic
oesophageal adenocarcinoma (OAC) and intra-mucosal cancer (IMC), of a material
which recognises, binds to or has affinity for certain polypeptides, or methylation of
certain nucleic acid sequences, wherein the polypeptides and/ or nucleic acid sequences
are as defined in any of claims 1to 16.
21. Use according to claim 20 of a combination of materials, each of which
respectively recognises, binds to or has affinity for one or more of said polypeptide(s) or
nucleic acid sequences.
22. An assay device for use in aiding detection of a surface abnormality in the
oesophagus of a subject, wherein said surface abnormality is selected from the group
consisting of low-grade dysplasia (LGD), high-grade dysplasia (HGD), asymptomatic
oesophageal adenocarcinoma (OAC) and intra-mucosal cancer (IMC), which comprises
a solid substrate having a location containing a material, which recognises, binds to or
has affinity for certain polypeptides, or methylation of certain nucleic acid sequences,
wherein the polypeptides and/ or nucleic acid sequences are as defined in any of claims
l to 16.
23. Akit comprising reagents for determining the expression level of each of
(i) p53;
(ii) c-Myc;
(iii) AURKA;
in a biological sample, and optionally further comprising reagents for determining the
methylation of MyoD and Runx3.
24. Amethod for aiding the detection of a surface abnormality in the oesophagus of
a subject, wherein said surface abnormality is selected from the group consisting of
low-grade dysplasia (LGD), high-grade dysplasia (HGD), asymptomatic oesophageal
adenocarcinoma (OAC) and intra-mucosal cancer (IMC), the method comprising
providing a sample of cells from said subject, wherein said sample comprises cells
collected from the surface of the subject's oesophagus, assaying said cells for TFF3,
wherein if TFF3 is detected in cell(s) of the sample, the method according to any of
claims 1to 16 is carried out, wherein detection of abnormal levels of at least one marker
in addition to detection of TFF3 indicates an increased likelihood of a surface
abnormality in the oesophagus of said subject.
25. Amethod according to claim 24 wherein detection of abnormal levels of at least
two markers in addition to detection of TFF3, preferably least three markers in addition
to detection of TFF3, preferably least four markers in addition to detection of TFF3,
preferably all five markers in addition to detection of TFF3, indicates an increased
likelihood of a surface abnormality in the oesophagus of said subject.
26. A method according to claim 24 or claim 25, wherein said cells are collected by
unbiased sampling of the surface of the oesophagus.
27. Amethod according to claim 26, wherein said cells are collected using a capsule
sponge.
28. Amethod for aiding the detection of a surface abnormality in the oesophagus of
a subject, wherein said surface abnormality is oesophageal adenocarcinoma (OAC), the
method comprising providing a sample of cells from said subject, wherein said sample
comprises cells collected from the surface of the subject's oesophagus, assaying said
cells for SMAD4, wherein if SMAD4 is detected in cell(s) of the sample an increased
likelihood of oesophageal adenocarcinoma (OAC) in the oesophagus of said subject is
indicated.
29. Amethod substantially as described herein.
30. Amethod substantially as described herein with reference to the drawings.