Cell Sampling Device
Field of the Invention
5 The invention relates to a cell collection device. In particular, the invention relates to a
cell collection device for sampling cells lining the oesophagus.
Background to the Invention
10 Certain cell collection devices are known. In particular, d capsule sponge type cell
collection device is described in PCT patent application number PCT/GB2006/003913.
Such a device comprises an abrasive material capable of collecting cells from the
surface of the oesophagus, together with means for its retrieval from the patient.
Typically such devices are swallowable.
15
Known devices typically operate by swallowing the abrasive material in a compressed
or stowed format. Upon reaching the stomach cavity, the material retaining the
device in a compressed or stowed format is dissolved or weakened, permitting
expansion of the compressed material back to its original size. Following this stage the
20 device is then retrieved by physically pulling it from the subject's mouth. This pulling
causes the device to travel out of the stomach cavity back up through the
oesophagus and out through the patient's buccal cavity and mouth. In travelling
along the subject's oesophagus, cells from the oesophageal lining are collected in the
abrasive material part of the device. These cells are subsequently analysed to aid in
25 the diagnosis or prognosis for the subject.
One known cell sampling device as described above is referred to as a capsule
sponge. This device comprises a compressible sponge like material. This is typically
attached to a cotton thread. The device is then compressed into a swallowable form,
30 such as by incorporation into a gelatine capsule, Such known devices have typically
had a cotton cord attached as the means of retrieval. This is a problem since cotton
cords can shed fibres inside the subject. Moreover, cotton cord of thus type can be
too rough. In addition, material may detach from the cord during use. Furthermore,
the known device has suffered from the problem of loss inside the subject being
35 sampled. This has typically occurred via separation of the uncompressed device from
the cord for its retrieval. Furthermore, the known devices can be difficult to swallow
due to friction of the cord on the oropharynx.
WO 2011/058316 PCT/GB2010/002077
Thus, known cell sampling devices suffer from a range of problems and drawbacks. The
present invention seeks to overcome problems associated with the prior art.
Summary of the Invention
5
The present inventors provide improvement on the known cell collection devices. The
inventors have employed different cords or threads attached to the compressible
abrasive material of the device in order to improve swatlowability and ease of use of
the device. Furthermore, the inventors have designed a new attachment system for
10 the cord attachment to the abrasive material. This attachment system comprises
certain specific classes of knot that provide superior strength. Furthermore, the knots
used are advantageously shown to reduce losses of the device in the subjects during
use. Thus, the invention provides a stronger and safer cell sampling device. The device
also enjoys benefits of superior swallowability and ease of use/retrieval.
15
Thus in one aspect the invention provides a swallowable cell sampling device
comprising an abrasive material capable of collecting cells from the surface of the
oesophagus, and a means for retrieval wherein the means for retrieval comprises a
cord
20 characterised in that the cord is attached to the abrasive material by means of a hitch
knot.
Suitably said hitch knot is a double overhand knot.
25 Suitably said abrasive material is compressible.
Suitably said abrasive material comprises reticulated polyurethane.
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 the hitch knot.
30 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
35 at 30 degrees Celsius.
Suitably the device comprises an unswallowable element at the end distal from the
swallowable abrasive material.
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In another aspect, the invention relates to a kit comprising a device according to any
preceding claim, and reagent for use in the detection of a cellular marker,
5 In another aspect, the invention relates to a method for aiding the diagnosis of Barrett's
oesophagus or Barrett's associated dysplasia In a subject, said method comprising
sampling the cellular surface of the oesophagus of said subject with a device as
described above, and assaying the cells for a cellular marker, wherein detection of
such a marker indicates increased likelihood of the presence of Barrett's or Barrett's
10 associated dysplasia.
A kit as described above or a method as described above wherein the marker is
selected from the group presented in Table 1.
15 A kit as described above or a method as described above wherein the marker is
selected from the group presented in Table 2,
A kit as described above or a method as described above wherein the marker is
selected from the group presented in Table 3.
20
Detailed Description of the Invention
A preferred embodiment of the invention is a 'capsule sponge' device where the
abrasive material comprises a sponge or sponge like material such as polyurethane
25 mesh and wherein said material is packed or compressed into a gelatine capsule for
ease of swallowing. Thus the generic term 'sponge' or 'capsule sponge' is sometimes
used to discuss the device of the invention for ease of understanding but it will be
apparent that other embodiments of the invention are envisaged and that the
invention is not to be understood as limited only to preferred capsule sponge
30 embodiment(s).
The term 'comprises' (comprise, comprising) should be understood to have its normal
meaning in the art, i.e. that the stated feature or group of features is included, but that
the term does not exclude any other stated feature or group of features from also
35 being present.
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Retrieval
The device of the invention suitably comprises means for retrieval from the subject
being sampled. Suitably the means for retrieval may comprise a cord. The cord is
5 occasionally referred to herein as thread/string.
The cord used should suitably be slippery. By slippery it is meant having a low coefficient
of friction, This has the advantage of easing the swallowing of the device. This
has the further advantage of easing retrieval of the device. A further advantage is to
10 reduce discomfort to the patient during use. By low co-efficient of friction is meant a
co-efficient of friction which is lower than that of a cotton cord of similar diameter.
The cord used should suitably be smooth. This has the advantage of being easier to
swallow. This has the further advantage of avoiding sticking to the mucosa in the
15 mouth, throat or oropharynx.
Dimensions
Suitably the cord or thread should be selected so as to avoid injury to the oropharynx of
20 the subject during use. Thus, in one aspect the invention relates to a new use of
surgical thread which has formerly been applied only as suture material. Suture
material is an example of a suitable cord for use in the present invention. Suitably the
thinnest possible cord is used, whilst remaining thick enough to avoid cutting the patient
during use,
25
Attention should be paid to choosing the correct thickness of the cord. Too thick a
cord can be difficult to swallow, yet too thin can cause laceration to the throat.
The cord should suitably be suitably thick to avoid cutting the patient during use. Thus
30 suitably the cord has a minimum diameter of at least 0.3mm.
The cord should be suitably thin in. order to minimise discomfort and improve
swallowability. Thus suitably the cord has a maximum diameter of 0.7mm.
35 Thus, the cord which may be used according to the present invention suitably
comprises a cord having a diameter in the range 0.3 to 0.7 mm, more suitably in the
range 0.4 to 0.6 mm. A most suitable cord diameter is approximately 0.5mm, most
suitably 0.5mm.
4
WO 2011/058316 PCT/G112010/002077 .
It should be noted that values given for cord diameters herein are of course subject to
usual measurement tolerances . In particular the figures given should be allowed a
tolerance of +/- 0.05 mm. Thus a size of 0.5 mm should be understood to embrace 0.45-
5 0.55 mm with the tolerance noted.
The length of the cord is matter for the operator . The cord needs to be long enough to
allow deployment of the device into the subject 's stomach cavity whilst retaining
enough cord extending into the buccal cavity or beyond to allow commencement of
10 retrieval. Thus the optimal cord length is the distance from the buccal cavity to the
stomach cavity plus an extra length such as 5 cm to permit grasping and retrieval of
the device. Longer cords may be advantageous to provide ease of withdrawal and/or
to provide reassurance to the subject.
15 Suitably the cord is at least 60cm long . Suitably the cord is less than 75cm long.
Suitably the cord is in the range 60-75cm , suitably 64 -69cm, suitably about 67cm long,
suitably 67cm long.
20 Strength
The cord must be chosen to have the strength necessary to prevent against breakage
upon withdrawal.
25 Suitably the cord is break resistant to a load of at least 2.4Kg , suitably to at least 3.2Kg,
suitably to at least 3.5Kg , suitably to at least 4.6Kg.
further Properties
30 Suitably the thread or cord used is of pale colour such as while . This has the advantage
of being preferred by subjects.
Suitably the abrasive material or capsule used is of dark colour such as black . This has
the advantage of being preferred by subjects during use . This has the further
35 advantage of disguising or making less prominent the appearance of any biopsy
material trapped on the abrasive part of the device once retrieved from the subject.
This can assist in reducing distress in the subject during operation.
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Suitably the cord comprises a cord suitable for internal use. Suitably the cord comprises
a cord certified for internal use. Certified means approved by a relevant health
authority such as the UK MHRA (Medicines and Healthcare products Regulatory
Agency). Suitably the cord should not shed fibres inside the subject. Suitably the cord
5 should display no loss of material from cord into the digestive system of the subject.
The cord may comprise surgical suture material.
At the non-cell-collecting end, suitably the cord is tethered to an anchor to prevent
10 swallowing. Suitably the anchor may comprise cardboard.
Attachment
The means for retrieval (such as a cord) must be attached to the cell sampling abrasive
15 material of the device. It is a key teaching of the present invention that a particular
knot system is used to attach the cord to the abrasive element of the device.
Suitably a double half knot should be used. An example of a particularly suitable
double half knot is a double overhand knot. This type of knot arrangement provides the
20 advantage that the first half knot falls onto or presses against the second half knot. This
is advantageous because the more force that is applied to the cord during retrieval,
the more the knot tends to tighten (rather than loosening).
An advantage of the knot system used in the invention is that the cord is attached to
25 the abrasive material via a loop. This loop may reduce in size with the force applied.
Clearly, reduction of the size of the loop will partially constrict or compress the internal
part of the abrasive material. Importantly however, this knot arrangement does not
significantly reduce the diameter of the abrasive material. In particular, this, loop
arrangement does not significantly decrease the lateral diameter of the abrasive
30 material. By lateral diameter is meant the diameter in the axis perpendicular to the
force applied by the cord during retrieval. This has the advantage that, although the
shape of the abrasive material may change slightly during constriction of the loop
under the force of retrieval, the lateral diameter of the abrasive material remains
substantially constant and therefore remains effective in sampling the cells of the
35 oesophagus during retrieval.
The knot attaching the cord to the sponge optimised for :
WO 2011 /058316 PCT/GB2010/002077
Numerous knots were investigated that for use with slippery cord material including
material which can become more slippery when wet e.g. when mixed with mucous
from the mouth or stomach.
5 Multiple knots may be used . Suitably only a single knot is used , which has the
advantage of simplifying construction of the device . Of course a single knot may
comprise multiple hitches or elements and refers to a single entire knot rather than a
single thread element within an individual knot.
10 Suitably the knot of the device has one or more of the following properties:
(i) The knot of the invention is suitably tightened under tension to prevent
detachment of the sponge from the cord upon removal.
IS (ii) The knot suitably does not affect significantly the external diameter of the
abrasive material such as sponge when under tension.
(iii) The knot suitably does not leave an overhang loop of cord material at the
bottom of the abrasive material such as sponge.
20
Suitably the knot of the device has two or more of said properties ; suitably the knot of
the device has all three of said properties.
Examples of classes of knot , and of specific knots, having these properties are discussed
25 below.
Two half hitch knot
The two half-hitches is a type of knot , specifically a binding knot or hitch knot . It consists
30 of on overhand knot tied around a post, followed by a half-hitch. Equivalently, it
consists of a half-turn around a post followed by a clove hitch of the running end
around the standing part.
This knot is also sometimes referred to as a clove hitch over itself.
35
The person skilled in the art will be familiar with the standard knot names and
terminology used herein and will therefore be able to tie them without further
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guidance. Nevertheless, the following three-step process for tying the two half-hitches
is provided for ease of understanding: .
-Begin by forming a clockwise loop around the pole, with the working end of the rope
5 on top. Bring the working end through the loop. At this point, you have an overhand
knot around the pole.
-Bring the working end down and to the left. Loop it under the standing end. Pull the
working end through the loop just formed, tighten, and slide the knot along the
standing end up to the post.
10 -A correctly tied two halt hitches resembles a clove hitch tied around the standing end
of the line, not a cow hitch.
In discussion of knots presented herein, the context of the device must be borne in
mind. For example, there is clearly no 'pole' incorporated into the device of the
15 invention. The 'pole' is merely a common point of explanation for knot tying and in the
context of the invention should be understood to be the material around which the
knot is being tied, such as the centre of core of the abrasive material (when the cord
suitably runs under the surface of the material therefore passing around the inner part
of the abrasive material collector part of the device.)
20
Alternate Knots
Suitably one or more of the following knots may be used for attachment:
Anchor hitch knot (this is an alteration in hitch knot).
25
Suitably the knot is a hitch knot.
All types of hitch knots can potentially be used for attachment, except single hitch
knots which are not suitable. It will be borne in mind that some of the hitch knots are
30 complex and therefore are less desirable and/or less practical for manufacture.
Suitably the knot is a simple hitch knot, which has the advantage of ease of
manufacture.
Alternate Hitch knots
35 alternate ring hitching, anchor bend variant, bale sling hitch, barrel hitch, becket hitch,
blackwall hitch, blake's hitch, boom hitch, bottom loaded release hitch, buntline hitch,
cat's pow, chain hitch, clinging clara, clove hitch, continuous ring hitching, cow hitch
variant, cow hitch with toggle, cow hitch, double half hitches, Farrimond friction hitch,
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garda hitch, ground-line hitch, half hitch, halter hitch, highpoint hitch, highwayman's
hitch, hitching tie, icicle hitch, killick hitch, knute hitch, lighterman's hitch, magnus hitch,
marline hitching, marlinespike hitch, masthead knot, midshipman's hitch, munter hitch,
munter friction hitch, ossel hitch, palomar knot, pile hitch, prusik knot, reverse half
5 hitches, round hitch, round turn and two half hitches, sailor's gripping hitch, sailor's hitch,
Siberian hitch, single hitch, slippery hitch, Snell knot, snuggle hitch, taut-line hitch, timber
hitch, trilene knot, trucker's hitch, tugboat hitch, uni knot, wagoner's hitch
Most suitably said hitch knot is a double overhand knot. This has the advantage of
10 ease of tying, This knot has each of the properties (1) (if) and (iii) given above. Further
advantages may be apparent from the examples section of the application.
Double loop bow line knot is suitably not used in the invention, suitably the attachment
does not comprise a double loop bow line knot.
15
Known devices have not used the knot systems described herein. Known devices have
only used full knots rather than half knots. In particular, known devices have used
multiple "granny knots" and have not used the slidable noose system for attachment as
described herein.
20
Free End
The free end of the cord refers to the free part after the knot at the cell collector end of
the cord. Suitably the free end is at least 1 cm. Suitably the free end is in the range 1.0-
25 2.0cm. Suitably the free end may be at least 1.5cm, suitably at least 1.7cm, suitably
2.0cm.
Problematic knots
30 Unsuitable knots include a Sheet Bend - this knot should not be used as it is slippery and
may not withstand the pulling force on the cord; Double sheet bend should not be
used as it is slippery and may not withstand the pulling force on the cord and can also
be complex to perform; Reef Knot should not be used on slippery threads/cords; Clove
hitch is a knot for a looping thread or cord, not for a single free end thread or cord, and
35 leaves two threads at the free end which is not acceptable for the device of the
invention and so suitably the clove hitch should not be used. In addition to the above,
it should be noted that a Bow Line knot is slippery; structure of this knot is similar to a
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WO 2011/058316 PCT/GB2010/002077
sheet bend; thus the bow line knot is suitably avoided. Thus suitably the attachment to
the abrasive material does not comprise a knot mentioned in this paragraph.
Attachment at non-swallowed end
5
It will be noted that the discussion of attachment and knots refers to the joining of the
abrasive material to the cord. The non-abrasive-material end of the cord (i.e. the nonswallowed
end) may be joined to a further element for example to prevent accidental
swallowing, to facilitate withdrawal or for any other purpose. The joining of the cord at
10 the non-swallowed end may be by any suitable means such as welding, stitching,
stapling, weaving, gluing or any other method including knotting but when joined by
knotting the knot may be any suitable knot for secure fastening and need not be
restricted in the manner described as part of the invention for joining of the cord to the
abrasive material.
15
Abrasive Material/Capsule
The abrasive material suitably comprises a sponge or sponge like material.
20 The qualities of the abrasive material are discussed in more detail below. A key feature
is that the material needs to be abrasive enough to collect as many cells as possible
whilst at the same time avoiding damage to the oesophagal lining. These
advantageous features may be achieved for example via use of a sponge or
honeycomb form of abrasive material. This porous or cavitated form of material
25 maximises collection and/or entrapment of cells inside and on the surface of material.
Moreover, the cavities or hollows in sponge like or honeycomb material such as
reticulated polyurethane also facilitate compression which is advantageous in reducing
the size of the material at administration for example via a soluble capsule.
30 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.
35
Suitably the material is dimensioned to fit into a swallowable capsule such as a gelatine
capsule, suitably. in a compressed or stowed form. Suitably said material does not
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WO 2011 /058316 PCT/GB2010/002077
sampling techniques such as biopsy collection techniques which penetrate below the
surface of the oesophagus.
In another aspect , the invention relates to a non -invasive method for aiding the
5 diagnosis of Barrett's oesophagus or Barrett 's associated dysplasia , comprising assaying
cells from the surface of a subject's oesophagus for a cellular marker , wherein
detection of such a marker indicates increased likelihood of the presence of Barrett's or
Barrett's associated dysplasia. In this embodiment , preferably the actual sampling of
the cells is not part of the method of the invention.
10
In another aspect, the invention relates to a method for aiding the diagnosis of
squamous cell carcinoma in a subject , said method comprising sampling the cellular
surface of the oesophagus of said subject, and assaying the cells for a cellular marker,
wherein detection of such a marker indicates increased likelihood of the presence of
15 squamous cell carcinoma . Preferably said sampling is not directed to a particular site
within the oesophagus. Preferably only the surface of the oesophagus is sampled. This
has the advantage of avoiding more invasive sampling techniques such as biopsy
collection techniques which penetrate below the surface of the oesophagus.
20 In another aspect, the invention relates to a non -invasive method for aiding the
diagnosis of squamous cell carcinoma , comprising assaying cells from the surface of a
subject ' s oesophagus for a cellular marker, wherein detection of such a marker
indicates increased likelihood of the presence of squamous cell carcinoma . In this
embodiment, preferably the actual sampling of the cells is not part of the method of
25 the invention.
Preferably the method of the invention is conducted in vitro.
Preferably for Barrett ' s oesophagus or Barrett 's associated dysplasia the marker is a non-
30 squamous cellular marker.
Preferably the marker is a marker of cellular proliferation. This is particularly preferred for
squamous cell carcinoma embodiments of the invention.
35 Preferably the marker is a marker of columnar cells.
In another aspect , the invention provides a method as described above wherein
sampling the cellular surface of the oesophagus comprises the steps of
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(i) introducing a swallowable device as described above into the subject,
(ii) retrieving said device by withdrawal through the oesophagus, and
(iii) collecting the cells from the device.
5 Preferably step (i) comprises introducing a swallowable device as described above into
the subject's stomach.
Mills
10 In another aspect, the invention provides a kit comprising a device as described
above. Suitably said kit further comprises a local anaesthetic. Preferably said local
anaesthetic is a spray or lozenge, preferably a spray.
In another aspect, the invention provides a kit as described above further comprising a
15 container for receiving said swallowable device after withdrawal, said cohtainer having
a quantity of preservative fluid therein. Preferably the container is a watertight
container. Preferably the preservative fluid is a cell preparation fluid. Preferably said
fluid is thin preparation fluid for production of slides for examination of the sampled
cells.
20
In another aspect, the invention provides a kit as described above wherein said device
comprises a capsule sponge.
In another aspect, the invention provides a kit as described above wherein said
25 swallowable device comprises withdrawal means such as string or cord.
In another aspect, the invention provides a kit as described above further comprising a
device for severing said withdrawal means. Preferably said device comprises a blade
or scissors.
30
In another aspect, the invention provides a kit as described above further comprising a
container for administering drinkable fluid, such as water, to the subject.
In another aspect, the invention provides a kit as described above further comprising
35 gloves. These advantageously protect the sample from contamination upon
withdrawal of the device.
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Preferably said kit further comprises reagent for use in the defection of a cellular
marker.
In another aspect, the invention provides a kit as described above further comprising
5 reagents for use in the detection of at least one marker selected from the group
consisting of markers presented in Table 1, or the group consisting of markers presented
in Table 2, or the group consisting of markers presented in Table 3.
In another aspect, the invention provides a kit further comprising a watertight container
10 and preservative fluid. Preferably said fluid is for liquid based cytology, preferably said
fluid is commercially available thin preparation fluid for production of slides for
examination of the sampled cells.
In another aspect, the invention provides a kit as described above further comprising a
15 local anaesthetic spray or lozenge.
In another aspect, the invention provides use of a device as described above in the
diagnosis of Barrett's oesophagus or Barrett's associated dysplasia.
20 Barrett's Oesophagus and Dysplasia
Barrett's oesophagus can occur without dysplasia. Approximately 1% of patients with
Barrett's oesophagus will develop dysplasia each year. At any given time,
approximately 20% of patients with Barrett's oesophagus will have dysplasia. Cancer
such as adenocarcinoma develops from dysplasia and is regarded as one extreme
25 form of dysplasia, even though pathologically the conditions clearly differ.
Adenocarcinoma is regarded as one extreme form of dysplasia, and its defection and
diagnosis is discussed herein.
Thus it can be appreciated that the invention may be applied to detection and
30 diagnosis of a single progressive disease state that has recognisable discrete stages.
These stages comprise Barrett's oesophagus, Barrett's oesophagus associated dysplasia
including adenocarcinoma, which arises therefrom.
The normal state of the cells in the oesophagus is that of squamous epithelium. In
35 Barrett's oesophagus, these cells take on the characterisics of columnar epithelium and
undergo further changes as they progress through the disease states outlined above.
Thus, non-squamous cells in the oesophagus are abnormal and correlate with Barrett's
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oesophagus.and potentially with dysplasia and more serious abnormalities as discussed
herein.
Surface Sampling and Techniques
5 The device described facilitates sampling of the cells 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.
Preferably substantially the entire surface of the oesophagus is sampled, preferably the
10 entire surface e.g. the complete inner lumen.
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. Optimally
15 the abrasive material needs to be abrasive enough to collect as many cells as possible,
without causing damage to a subject's oesophageal lining. 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
20 removed from the oesophagus.
The material 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
adenocorcinoma cells present. In a most preferred embodiment, preferably the
25 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
30 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,
35 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
15
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approximating its uncompressed size. Alternatively it may be a material which expands
eg. 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
5 ' 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
10 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.
15 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 sponge comprises polyurethane such as polyurethane sponge,
20 preferably reticulated polyurethane.
Preferably the capsule coat is at least partially formed from gelatine. Preferably the
capsule coat is fully formed from gelatine.
25 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.
30
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
35 a partial coat of soluble and/or digestible material such as gelatine.
Preferably the expanded (eg. decompressed) abrasive material of the device is
approximately 3cm in the plane perpendicular to the axis of the oesophagus.
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Preferably this is the approximate diameter of the oesophageal lumen. More
preferably this is slightly larger than the diameter of the oesophageal lumen,
advantageously ensuring good contact with the inner surface of same as
withdrawal/sampling takes place.
5
It is a feature of the invention that the sampling is not directed eg. visually directed to
any particular part of the oesophagus. 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
10 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 even when the
method of the invention does not include collection of the sample.
15 Screening and Surveillance
Screening aspects of the invention relate to the detection and/or diagnosis of Barrett's
oesophagus. Typically in screening embodiments of the invention, the subjects being
examined, or from which the sample(s) are (or were) obtained, are of unknown status
for Barrett's.
20
Surveillance aspects of the invention relate to the detection and/or diagnosis of
dysplasia, including adenocarcinoma. Although clearly dysplasia and
adenocarcinoma are pathologically different conditions, adenocarcinoma can be
regarded as one extreme form of dysplasia. As is discussed below, the invention may
25 be advantageously applied to distinguish adenocarcinomo from dysplosia, depending
upon the molecular markers used. However, in general the discussion of surveillance
aspects of the invention relates to the detection of dysplasia, including
adenocarcinomo. Typically in surveillance embodiments of the invention, the subjects
being examined, or from which the sample(s) are obtained, are of unknown status for
30 dysplasia but will typically be known to have Barrett's.
In principle the difference between screening and surveillance aspects is of little
practical consequence to the working of the invention. The difference relates only to
the markers chosen. The sampling and combination aspects remain the some
35 between screening and surveillance. Indeed, it may be advantageous to combine
screening and surveillance ie. to examine cell samples for markers of Barrett's as well as
dysplasia including adenocarcinoma at the some time, thereby increasing the value of
the information obtained and achieving a more robust combined diagnostic output.
17
WO 2011/058316 PCT/G132010/002077
Markers
Markers that can be applied for Barrett's screening and surveillance are any markers
which are not expressed in normal oesophageal tissue, preferably any markers which or
5 not expressed in normal oesophagal surface cells.
Markers may be detected via nucleic acid based techniques (e.g. detection of
expression by RNA detection) or by protein based techniques (e.g. immunochemistry
using one or more antibodies recognising the polypeptide of interest; antibodies may
10 be easily raised against a marker gene of interest for example by making recombinant
protein and immunising a suitable host such as a rabbit or mouse). Some markers such
as Alcian blue are in fact vital dyes (histochemical stains) and so are simply assayed
directly.
15 For screening aspects fie. for detection of Barrett's oesophagus), preferably markers
that distinguish between intestinal metaplosia (Barrett's) and squamous oesophageal
cells or gastric cardia are used. These markers include markers of epithelial
differentiation.
20 The use of columnar markers is particularly preferred. The technical benefit of using
columnar markers is that only columnar cells are detected by using them. This means
that squamous cells (whether normal or cancerous) are not stained by columnar
markers. This is an advantage because Barrett's cells and dysplastic cells arising
therefrom such as adenocarcinoma cells are columnar and can thus be selectively
25 identified by use of columnar marker(s). This advantageously improves signal and also
reduces background and alleviates the need to apply further distinguishing markers,
thereby simplifying the procedure by directly detecting columnar cells in this manner.
Any other markers known to be differentially expressed in Barrett's versus normal
30 oesophageal surface cells may be employed.
Alternative markers may be identified using an expression microarroy comparing gastric
cardia and squamous cell biopsies. Any marker which is differentially present in these
cell types may be used in the present invention.
35
For surveillance aspects, preferably markers whose expression correlates with the
degree of dysplasia are used. Preferably such markers are used for the stratification of
patients of risk. Preferably such markers include proliferation markers such as K167 and
18
WO 2011/058316 PCT/GB2010/002077
Mcm proteins, proliferation and DNA damage markers such as PCNA, cyclins such as
cyclin D and/or cyclin A. aberrant p53 for example p53 LOH, p53 mutation, or p53
overexpression such as immunohistochemical detection thereof, p16 loss including
methylation, and aneuploidy for example measured by flow cytometry or image
5 cytometry. In slightly more detail, growth factors (such as EGF), growth factor receptors
(such as EGFR) as well as cytokines (IL-4) and molecules involved in inflammatory
response (COX-2) were shown to have an aberrant expression in BE and subsequent
progression to AC, and are therefore useful markers according to the present invention.
In vitro and ex vivo work has shown that acid and bile stimulation induced DNA
10 damage, MAP kinase pathway and the NFKB pathway and decreased apoptosis
therefore markers involved in the detection of DNA mutation and damage (e.g. ATM,
ATR), markers of apoptosis (p53) and markers from the MAPK pathway (erk, p38) and
markers from the NFKB are useful. Furthermore, bile acids increase the retinoic acid
pathway (CYP26Ai, RAR) which is linked to the induction of metaplasia in chick embryo
15 oesophagus. A number of other pathways have been involved in the development of
BE and progression to cancer such as TGF(1 and BMP pathways.
Indeed, any marker known to correlate with the degree of dysplasia would be suitable,
including many oncogenes and tumour suppressor genes. In particular, markers
20 mentioned in Fitzgerald RC Clin Gastroenterol Hepatol Complex diseases in
gastroenterology and hepatotogy: GERD, Barrett's, and esophageal adenocarcinoma.
2005, 3:529-37 or in Fitzgerald RC Recent Results in Cancer Res Genetics and prevention
of oesophageal' adenocarcinoma 2005, 166:35-46 may be suitable for use in the
present invention.
25
Most suitable markers according to the present invention are now discussed.
We describe genes with potential as biomarkers for use in the invention for example in
analysing cells harvested with capsule sponge.
30 Thus we disclose biomarkers for detection of Barrett's oesophagus. The following table
gives a breakdown of the number of genes at each stage of the experimental process.
The further in the process the better marker a particular gene is likely to be.
19
WO 2011/058316
Process/Type Public dataset
PCT/GB2010/002077
In house dataset
Dysregulated genes 18 191
Token to PCR 18 20
Validated by PCR 3 9
5
Taken to immunohistochemistry 3 Ongoing
Validated by immunohistochemistry 2 N/A
Genes taken to capsule sponge 2 N/A
The 20 genes from the in house datasets taken to the PCR were selected according to:
10 -High level of expression and high statistical significance
-Presence of suitable antibodies
Specific markers are now discussed in more detail:
15 Table 1 : 3 markers currently used wHh capsule sponge
Marker (e.g. Details e.g. Gene bank Notes
gene) name accession number(s)
Not a gene target but an
histochemical staining
Atcian blue technique
Mcm 2 may be
used for squamous
Mcm2 NM004526 cell carcinoma
TFF3 NM_003226
Table 2: 11 Markers validated at the PCR level. (Validation of the protein level may be
carried out If desired.)
Marker name Details e.g. Gene bank accession number(s)
ABP1 NM_001091
DDC NM_000790 NM_001082971
HOXC10 NM_017409
KCNE3 NM_005472
LAMC2 NM_005562 NM_018891
MUC13 NM_033049
MUC17 NM_001040105
20
WO 2011 /058316 PCT/GB2010/002077
NMUR2 NM_020167
PIGR NM002644
TSPANI NM_005727
HOXBS NM_002147
Table 3: 161 Genes differentially expressed between Barrett 's vs normal oesophagus
and gastric cardla. The genes are ordered from the most advantageous to the least
preferred (highest statistical significance and expression level to lowest significance and
5 expression level).
GeneSymbol Gene bank accession number(s)
RNF217 NM_152553
CCL28 NM_148672
AGR3 NM_176813
CFTR NM_000492
PAQR5 NM_001104554 NM_017705
BNIP3 NM_004052
GOLM1 NM_177937
PLA2G10 NM_003561
KCNK5 NM_003740
MLSTDI NM_018099
SLC 16A7 NM_004731
NFE2L2 NM_006164
CGNLI NM_032866
CALML4 NM_001031733 NM_033429
ACSL5 NM_016234 NM_203380 NM_203379
KRT8 NM_002273
TMC7 NM_024847
FAT NM_005245
CES3 NM_024922
SLC7A7 NM_003982
REG4 NM_032044
CATSPERB NM_024764
TSPAN3 NM_005724 NM-) 98902
SLC37AI NM_018964
21
CVO 2011/058316 PCTIGB2010/002077
GPRC5A NM_003979
GPT2 NM_133443
PAIP2B NM_020459
TRIM29 NM_012101
IL18 NM_001562
HSDUBI I NM 016245
GSDML NM001042471 NM_018530
TACSTDI NM 002354
HSD17B2 NM002153
KRT7 NM_005556
CLIC6 NM_053277
ATP2C2 NM_014861
HEPH NM_014799 NM_138737
TPD52L1 NM_003287 NM_001003395 NM001003396 NM_001003397
HOXB6 NM 018952
PLSI NM_002670
ILURN NM_173841 NM_173842 NM173843 NM_000577
NJ5E NM 002526
CAB39L NM_001079670 NM 030925
S100AI4 NM_020672
GDA NM_004293
TRIM31 NM_007028
ARPCIB NM_005720
SLC 16A l NM_003051
TMCS NM_024780 NM_001105248 NM_001105249
CPEB2 NM_182646 NM_182485
LOC93432 ENST00000397504
F5 NM_000130
VLDLR NM_003383 NM_001018056
GCNT3 NM_004751
MBOAT2 NM_138799
CPS) NM_001875
GALM NM_138801
DGKD NM_152879 NM_003648
FAM 1028 NM_001010883
22
WO 2011/058316 PCT/GB2010/002077
LYN NM_002350
SFN NM_006142
GALNT7 NM017423
EMPI NM_001423
CSTB NM_000100
RHOC NM_001042678 NM- 175744 NM_001042679
FLJ14959 AK027865
SNRPN NR_001294
ANKS48 NM_145865
PCLKC NM 017675
ADH7 NM_000673
LYZ NM_000239
S100A16 NM_080388
SLC6A20 NM_020208 NM022405
SCNNIG NM_001039
HKDC1 NM_025130
SLC7A2 NM_001008539 NM_003046
SPG20 NM_015087
37681 NM 178450
FGFBPI NM 005130
CA9 NM_001216
RDX NM_002906
SAMD9 NM_017654
SERPINB5 NM_002639
NMU NM_006681
CLRN3 NM_15231 I
SLC9A4 NM_001011552
VTCNI NM_024626
LOC339977 NM 001024611
FUT9 NM_006581
GALNT5 NM_014568
NR5A2 NM_205860 NM_003822
OLFM4 NM_006418
LY75 NM_002349
23
WO 2011/058316
SCPEPI
TACSTD2
MYO1A
BTNL8
VIL1
SLC28A2
DPP4
AZGPI
CDH17
NPNT
ALDHIAI
ATP13A4
ATP7B
IL2RG
POSTN
FCGBP
GPA33
DSC2
COL6A3
VNN1
SLPI
AIM1
PRKAA2
GUCY2C
P13
TIMP 1
APOL1
ANPEP
SLC34A2
DMBTI
RGS2
PAPSS2
BCMO 1
ADH6
PCT/GB2010/002077
NM_021626
NM_002353
NM005379
NM024850 NM001040462
NM_007127
NM_004212
NM_001935
NM_001185
NM_004063
NM_001033047
NM_000689
NM_032279
NM_000053 NM_001005918
NM 000206
NM 006475
NM_003890
NM_005814
NM_024422 NM_004949
NM057167 NM_057165 NM_057164 NM_004369
NM004666
NM_003064
NM_001624
NM_006252
NM004963
NM_002638
NM 003254
NM_003661 NM_ 145343
NM_001150
NM006424
NM_007329 NM004406 NM017579
NM_002923
NM_004670 NM_001015880
NM_017429
NM_000672 NM_001102470
24
CVO 2011/058316
TM4SF20
CHSTS
HHLA2
FABPI
SNORD1 16-
21
MYO7B
MIA
MEPIA
SLC3AI
PLAC8
TFPI
PGC
MUC2
LIPF
FABP2
SI
SLC17A4
GSTA1
PDZKI
RAB36
REGIA
SPINK4
CXCL l
GKNI
BTNL3
ADH4
ALDOB
CXCL2
SLC26A3
MMP7
UPKIB
MEP1B
CAI
PRSS7
NM_024795
NM_024533
NM_007072
NM_001443
NR_003335 NR 003106
NM_001080527
NM_006533
NM_005588
NM_000341
NM 016619
NM_006287 NM_001032281
NM_002630
ENST00000361558
NM_004190
NM_000134
NM_001041
NM_005495
NM_145740
NM_002614
NM_002867
NM_002909
NM_014471
NM_001511
NM_019617
NM_197975
NM_000670
NM_000035
NM_002089
NM_0001 I I
NM_002423
NM_006952
NM_005925
NM_001738
NM_002772
25
PCT/GB2010/002077
'NO 2011/058316 PCT/GB2010/002077
Most suitably all markers shown above may be used (e.g. 161 from table 3; plus I I from
table 2; plus 3 from table 1). This has the advantage of maximising statistical
significance and eliminating any potential artefacts in the results. However, assessing
5 this quantity of markers may become impractical or indeed unnecessary for many
applications. Thus, suitably up to I I markers are assayed.
Clearly the number of markers assayed will depend on the format or mode chosen by
the operator for analysis. In a primary care setting the emphasis may be on simplicity
10 and/or avoidance of use of specialist equipment and so the number of markers may
be minimised in such settings-for example to three or fewer markers of Table 1.
When an array such as a nucleic acid array is used to analyse the markers, it is very
straightforward to analyse multiple markers on a single array or chip. Thus for these
15 modes of analysis, greater numbers of markers may be used such as 50, 100, 150, 161 or
even more.
Multiplex PCR may be used to assay the markers. In this embodiment suitably up to 20
markers may be analysed in the some procedure.
20
Most suitably markers used are one or more of those shown in table I; more suitably two
or more of those shown in table 1; most suitably each of those shown in table 1.
Most suitably Trefoil factor 3 (TFF3) is a marker used in the present invention . TFF3 and its
25 use is described in W02005/013802.
Marker Assay/Detection
Assaying for a marker means determining the presence or absence of said marker.
Preferably assaying means immunological staining or visualisation of the marker.
30
Marker expression (marker gene expression) may be detected by any suitable means
known to those skilled in the art. Expression may be detected at the nucleic acid or
protein level. Expression may be by mass spectrometry and assignment of the mass
readouts to particular protein moieties. At the nucleic acid level, detection is
35 preferably by monitoring of mRNA levels. Preferably expression is detected at the
protein level. Preferably marker gene expression refers to marker protein expression.
Preferably marker protein expression is determined by direct or indirect detection of
marker protein. Preferably such protein is detected by immunochemical means.
26
WO 2011/058316 PCTIGB2010/002077
Preferably the marker protein is detected by an antibody capable of reacting with that
protein, and subsequent visualisation of said antibody. Preferably the antibody is a
polyclonal antibody or a monoclonal antibody. Preferably when the antibody is a
polyclonal antibody it is an immunopurified polyclonal antibody. Preferably the
5 antibody is a monoclonal antibody. Use of secondary and even tertiary or further
antibodies may advantageously be employed in order to amplify the signal and
facilitate detection. Preferably marker protein(s) are visualised by use of
immunohistochemicat means, such as immunoftuorescent means, directly or indirectly
bound to the marker protein(s). Preferably detection is by antibody to the marker.
10
Other suitable assays include ELISA - fluorescent in-situ hybridisation of fish and FACS -
fluorescence analysis of cell sorting.
Sample
t5 It will be appreciated that the sample preferably comprises a population of individual
cells obtained by the sampling procedures described herein. Thus, the detection of the
markers preferably refers to detection of the markers in at least one cell within said
population of cells. The detection of an appropriate marker in any cells in the sample
will be indicative of Barrett's or a Barrett's associated dysplasia. The absence of any
20 cells showing the marker from the population of cells of the sample will be indicative of
lack of Barrett's or Barrett's associated dysplasia. The proportion of cells showing
expression of the marker is less important. The proportion of cells showing expression of
the marker would not usually make a contribution to the diagnosis. The present
invention is based on the detection of any cell(s) showing the marker in the sampled
25 cell population, or the apparent absence of any cells showing the marker. In some
embodiments, it may be advantageous to determine the relative proportions of the cell
types or the proportion of cells displaying proliferative markers, as an optional step
dependent on the needs of the operator. However, for most embodiments of the
invention, the result will be expressed as a positive or negative, and the relative
30 proportions of cells will normally not be taken into consideration.
KHs
The kits of the invention are designed to provide for conducting the methods of the
35 present invention. Thus, the description of elements required for the methods of the
invention applies equally to the contents of the kits of the invention, which preferably
contain the elements required for practice of said methods. In particular, preferably
the kits contain reagent for detection of the marker or markers being used.
27
WO 2011 /05831 6 PCT/GB2010/002077
Preferably the. kit of the invention also contains a local anaesthetic for use in the
oesophagus. Preferably this may be in the form of a spray or lozenge, preferably a
spray.
5
Preferably the kit of the invention also contains a container for holding the device once
withdrawn from the subject. Preferably this container is watertight. Preferably the
container contains a preservative fluid. Preferably the container contains a liquid
based cytology fluid such as commercial thin preparation fluid for producing slides of
10 the sampled cells. Preferably the thin preparation fluid comprises a preservative.
Preferably the swallowable device is lubricated to aid swallowing, preferably the
withdrawal means is also lubricated. Thus, preferably the kit comprises lubricant.
15 Preferably the kit comprises a drinkable solution to aid swallowing the device.
Preferably said solution is flavoured to disguise the taste of the device, or to render it
more palatable. Preferably said solution is thickened eg. by addition of sugar or pectin
or other agent giving rheologicat characteristics such as viscosity or thickness. The
advantage of this is that a more viscous or dense solution will be more effective at
20 aiding passage of the device through the oesophagus during swallowing,
In order to save weight/volume in kits, preferably the solution(s) supplied are supplied in
powdered farm such that the operator reconstitutes them before use eg. by adding
water. Preferably the kit comprises a container for reconstitution. Preferably said
25 container is graduated to facilitate measurement of the correct amount of fluid such as
water.
Preferably the swallowable device does not comprise animal product Is).
30 Preferably the kit comprises anti-emetic eg. in lozenge. solution or powdered form, to
suppress any urge to vomit during introduction and/or withdrawal of the device.
Preferably the kit may comprise antacid such as acid-neutralising compound(s), or such
as pharmaceutical antacid for inhibition of acid production/secretion In the stomach.
35 Advantageously this may be used to inhibit a burning sensation of acid carried up the
oesophagus from the stomach upon withdrawal of the device. Furthermore, this may
be advantageous in preservation of the cell samples obtained with said device.
28
WO 2011/058316 PCT/GB2010/002077
Preferably the preservative fluid contains antacid and/or is buffered to the desired pH
for preservation of the cell sample obtained.
In one embodiment the kit preferably comprises a local anaesthetic spray, a capsule
5 sponge, a pot containing prep liquid (e.g. ThinPrepTM PreservCytTM SolutionTM), a label
for the pot. and an instruction leaflet for a health care professional who administers the
sampling.
Preferably the kit further comprises gloves (for health care professional such as a nurse
10 removing the capsule from the subject).
15
Preferably the kit further comprises scissors to cut the withdrawal means (e.g. cord).
Preferably the kit further comprises a plastic cup (for subject to drink fluid e.g. water).
Preferably the kit further comprises an information leaflet for the subject/patient.
In another embodiment the invention relates to a self testing kit such as a dip-stick
formal kit whereby said stick comprises reagents for detection of markers according to
20 the present invention and wherein in use dipping the stick into the pool of sampled cell
material leads to a visualised readout of the markers according to the present
invention, thereby providing information capable of aiding diagnosis as set out herein.
The device comprises withdrawal means. Preferably this is a string or cord based
25 means. Preferably the withdrawal means is graduated so that the operator can
estimate when the device is, or is likely to be, in the stomach. Furthermore, the
graduations advantageously allow monitoring of withdrawal of the device and allow
for standardisation of the rate of withdrawal and for optimisation of sample collection.
30 Preferably the withdrawal means comprises an unswallowable element at the end
distal from the swallowable abrasive material. This advantageously prevents
accidental swallowing of the entire device, inhibiting or preventing its withdrawal.
Preferably this unswallowable element is detachable in case of emergency when it
may be safer to allow the entire device to be swallowed and passed through the
35 alimentary canal.
29
WO 2011/058316 PCT/GB2010/002077
Further Kit Features
In some embodiments, it is probable that there will be a multi-part kit to provide for
different elements in different settings . The discussion above is focussed on the
5 preferred aspects of the kit of the invention which is the primary core application e.g. in
screening for intial detection in a subject. However, it will be apparent to the skilled
person that the oesophagus surface sample may be analysed at a location different
from the initial primary care setting in which subject (s) are sampled . For example, the
cell(s) may be analysed in a laboratory separate from the primary care setting in which
10 the sample is collected . In this embodiment it is apparent that the invention may relate
to multi-part kit (s) having a primary care component as well as a read -out component
(or laboratory component ), or the invention may even relate to the readout/
laboratory component of the kit per se . In this example, the read -out (or
laboratory) component of the kit may comprise one or more of the following elements:
15
- Consumables such as non-gynaecological microscope slides , and/or nongynaecological
filters.
- Equipment such as ThinPrepT M 20D0 processor.
20
- Detection of abnormal pathology - for the detection of Barrett ' s oesophagus using
immunohistochemistry; System for automated immunostaining e.g. if the samples are
stained using the DakoCyomation Ltd ChemMateT M system.
25 The kit may further comprise one or more of the following detection consumables such
as Dako Autostainer reagents vial ; ChemMateT"r detection kit : ChemMateTM Peroxidase
blocking solution ; ChemMateTM antibody diluent ; Mcm2 antibody; Goat serum; Bovine
serum albumin; Haematoxylin and/or Coverslips.
30 The kit may further comprise equipment such as Dako autostainer slides processor
(53400 Dako autostainer).
In order to facilitate analysis of the samples , the kit may comprise visualisation means
such as a microscope (such as an automated microscope ) e.g. Olympus BX41 with X10,
35 X20 and/or X40 objectives.
30
WO 2011/058316 PCT/G82010/002077
Further Advantages/Applications
Once tissue architecture is lost as in surface sampling, cytologists can no longer tell cell
types such as squamous, columnar, Barrett's etc apart. Furthermore, observation of
inflammatory cells such as lymphocytes no longer contributes to the diagnosis since no
5 positional information can be gleaned from their observation. However,
advantageously the present invention overcomes this problem by employing
biomarkers to identify the cell types even when the histological information has been
lost.
10 Although it is preferred to assay the cells by distribution onto slides, it may be
advantageous to perform the assay in a different format such as ELISA or FACS or
FISH. Preferably the cells can be assayed in one or more of these format(s) directly
from the capsule sponge or washings therefrom, advantageously avoiding the need for a
slide format analysis. If a slide format analysis is required, preferably cells are
15 concentrated onto the slides to produce fewer slides for the same number of cells,
thereby saving costs. In one embodiment, preferably the cells from the capsule sponge
are collected and their protein extracted and tested for the marker(s), thereby alleviating
the need for whole cell staining.
20 Advantageously pore size on the preferred capsule sponge sampling device can be
varied to regulate the number of cells harvested. For example, by reducing pore size the
number of cells (and thus the number of slides needed) may be advantageously reduced.
In highly preferred embodiments, markers are chosen to detect high risk Barrett's. This
has the further advantage that surveillance ie. remonitoring of patients with Barrett's to
25 detect future dysplasia including adenocarcinoma may be reduced or rendered
unnecessary since in one step the Barrett's is detected and graded as high risk, so
subsequent treatment can be prescribed immediately without expensive surveillance,
and without the risk that during surveillance the patient will go on to develop more
dangerous lesions before detection.
30
It is an advantage of the present invention that false negatives are extremely rare. Some
false positives can occur, eg. detection of naturally proliferating cells such as closing a
wound incurred by swallowing an abrasive foodstuff such as a fruit stone. However, a
negative result from the tests and kits of the present invention is very reliable so that
31
WO 2011/058316 PCT/GB2010/002077
patients can be excluded from unnecessary follow up procedures and can receive robust
reassurance at an early stage when a negative result is obtained.
Since the methods and kits of the invention are simple and low in cost, a much wider
5 screening programme can be undertaken for the same net cost to the service provider.
Preferably the tests of the present invention are carried out on a given subject at 3 year
intervals.
IO Another advantage of the invention is that the first signs of dysplasia can be very small
and may be missed by visual inspection or endoscopic biopsy sampling, but will be
detected according to the present invention. Similarly, 40% of subjects with high grade
dysplasia already have the cancer present. The present invention advantageously
allows better detection/diagnosis of these patients.
15
Use of sponge material as the abrasive material has the advantage of being able to
collect cells throughout its structure due to its preferred mesh construction, rather than
being limited to collection on the cell surface. This has the advantage of increased
yields.
20
Further Applications
Suitably the device of the invention may be applied for sampling of squamous cells
such as in aiding the diagnosis or prognosis of squamous carcinoma. This application
25 may be as well as, or as a separate application from, sampling of oesophageal cells
such as Barrett's oesophageal cells for aiding the diagnosis or prognosis of Barrett's
oesophagus, dysplasia, or adenocarcinoma.
Thus the device described herein finds application in squamous cell carcinoma -
30 suitably the biomarkers used will be chosen accordingly in this application of the
invention. For aiding the diagnosis or prognosis of squamous carcinoma, suitably the
markers used are markers of cellular proliferation. For squamous carcinoma
applications, Mcm2 is a most suitable biomarker.
35 The present invention will now be described, by way of example only, in which
reference will be made to the following figures:
32
WO 2011/058316 PCT/G132010/002077
Brief Description of the Figures
Figure I shows construction of device with cord under surface of abrasive material.
Figure 2 shows construction of device with cord under surface of abrasive material.
Figure 3 shows a photograph of an exemplary device of the invention.
5
Examples
Example I - Construction of device
An exemplary device has the following features or components
10
- abrasive material: Sponge (composition: reticulated polyurethane; density: 10 ppi;
shape: spherical; diameter: 3cm; supplier: Foam Conversion Ltd, Kempston, Bedforshire,
UK)
15 - means for retrieval: Cord (reference: White Force Fiber Code F500-W; size: No 2;
supplier: Teleflex Medical, USA)
- soluble capsule (reference: Gelatin Capsule; size: 00; supplier: Capsuline Inc., USA)
20 - unswallowable element: Cardboard retaining' the loose end of the cord (supplier:
Medical Wire & Equipment Ltd, UK)
The device is optionally packaged:
- Packaging (composition: sealed polythene bag; supplier: Medical Wire & Equipment
25 Ltd, UK)
The device may be irradiated with a minimum dose of 17 kGy, which is at a level
necessary to clean the device but not sterilise it. The device may be optionally
irradiated with a sterilising dose of radiation.
30
The knot used in tying the thread through and out of the capsule sponge:
Free end of the thread is passed in to the open sponge in such a way that the thread
forms a small loop with in the sponge just below the surface as shown in the Figure 1.
35
The free end of the thread is brought out and a loop is made using two overhand knots
(noose) one after the other (double overhand knot).
33
WO 2011/058316 PCT/GB2010/002077
Note that it is basically two overhand knots, with the second overhand knot acting as a
stop-knot.
The other free end of the thread is passed through this loop and pulled tight as in Figure
5 2.
At least Icm length is left at the free thread after the knot.
The cord runs under the surface of the sponge. The cord may optionally run through
10 deep in the centre of the sponge sphere.
The short loose end of the cord is at least I -centime tre-long.
The long loose end of the cord shall be wound round and attached to an
15 unswaltowable element which comprises a flat piece of cardboard (5-centimetre-long
and 3-centimeter-wide).
80 centimetres of cord shall be allowed for each sponge kit, including the knot.
20 The assembled device is then encapsulated into the soluble capsule.
Example 2: Further properties and features
Devices of the invention may be tested to check their performance and properties.
25
Devices may be tested as follows:
- measure the length of the cord from the end to the outer surface of the capsule - it
should be 60 centimetres minimum
30
- look for any breaks on the outside of the capsule - any breaks should be remedied
- calculate the time taken for the capsule to dissolve and the sponge to open in warm
water at 30 degree C - it should be 5 minutes maximum
35
- measure the size of the loop inside the sponge
- check the loop - should be just below the surface of the sponge
34
WO 2011/058316 PCT/GB2010/002077
break the capsule whilst compressed inside, but only deforms or breaks the capsule
once in use such as inside the subject.
5
10
Suitably the capsule has a uniform shape.
Suitably the capsule has a uniform size.
Suitably the capsule dissolves quickly such as dissolves in 30 degree centigrade water
within 5 minutes.
Suitably said capsule is intact all over and does not have any breaks and sharp ends.
This has the advantage of preventing injury while swallowing.
Further Advantageous Features
15
Suitably the device of the invention may be irradiated for cleanliness.
Suitably the device of the invention may be irradiated for sterility.
20 In a most preferred embodiment the device has the following properties:
-White colour cord
-Minimum length of cord - 60cros
-Cord smooth on the surface
-Cord loop inside the cell collecting abrasive material such as sponge - should loop just
25 below the surface
-Free end of the cord after the knot at cell collector end - minimum of 1 cm
-Attachment of cord to cell collector via Knot - Double hitch knot
-Cord Break resistant - Minimum requirement of 2.4kg
-Cord tethered to cardboard at non-cell-collector end to prevent swallowing
30
In another aspect, the invention relates to a method for aiding the diagnosis of Barrett's
oesophagus or Barrett's associated dysplasia in a subject, said method comprising
sampling the cellular surface of the oesophagus of said subject, and assaying the cells
for a cellular marker, wherein detection of such a marker indicates increased likelihood
35 of the presence of Barrett's or Barrett's associated dysplasia. Preferably said sampling is
not directed to a particular site within the oesophagus. Preferably only the surface of
the oesophagus is sampled. This has the advantage of avoiding more invasive
11
WO 2011/058316 PCT/GB2010/002077
- measure the free end of the cord after the knot - it should be I centimetre in length
minimum
5 - check that the knot complies with specifications set out herein
- measure the diameter of the sponge once the capsule dissolved after 5 minutes - it
should be 3 centimetres
10 - measure the weight the cord can hold before either tearing the sponge apart or the
thread getting undone from the sponge - the cord should hold 2.4 kg minimum
Example 3: ComparatIve Data
15 Devices according to the present invention were tested as above. Known (old)
devices were tested in parallel to demonstrate the technical advantages of the device
of the invention.
Exemplary features of a device according to the present invention include the
20 following:
Sponge:
Shape- round
25 Diameter- 3cm
Capsule:
Uniform shape and size
30 Not have any breaks and sharp ends
Dissolves in 30 degree centigrade water within 5 minutes
Retrieval means:
35 Cord
White colour thread
Minimum length of cord - 60cros
Smooth on the surface
35
WO 2011/058316 PCT/GB2010/002077
Loop inside the sponge - should loop just below the surface
Free end of the cord after the knot - minimum of I cm
Knot - Double hitch knot and complies with the specifications given
Break resistant - Minimum requirement of 2.4kg
5 Cord tethered to unswallowable element such as cardboard to prevent swallowing
Test performance of the known sponge (old sponge) in vitro:
Loop Fate when Sponge
Length Time taken
Free
Knot
Capsule inalde m". site
No of cord to open in end of spotlit with--
outside the tension when
In cm H2O at300 thread cation stood
pongo exceeded opened
larger
Thread
1 77,5 size, 6 min 11 sec Fine 0.7 cm Fine 1.42 kg 3cm
broke
Broken
Thread
2 75.5 Fine 4 min 54 sec Fine 0.8cm Fine 2.11 kg 3cm
broke
Thread
3 77 Fine 6 min 02 sec Small 0.8 cm Fine 3.10 kg 2.8cm
broke
Thread
4 77 Fine 6 min 29 sec Fine 0.8 cm Fine 2.51 kg 2.5cm
broke
Thread
5 77.5 Fine 4 min Fine 0.7 cm Fine 2.19 kg 3cm
broke
Thread
6 78 Fine 6 min 20 sec Fine 0.8 cm Fine 2.65 kg 2.8 cm
broke
Thread
7 78.5 Fine 6 min 29 sec Small 0.9 cm Fine 2.41 kg 2.5cm
broke
Thread
8 77 Fine 5 min 49 sec Fine 0.8 cm Fine 2.43 kg 3.1cm
broke
Thread
9 76 Fine 3 min 02 sec Fine 2 cm Fine 2,38 kg 2.8m
broke
Thread
10 78 Fine 4 min 32 sec Fine 2 cm Fine 2.41kg 2.8m
broke
10
Performance of the device of the invention (new sponge) in vitro:
Loop Fate when Sponge
Length Time taken Free Knol Weight
Capsule Inside maximum site
No of cord to open In end of specifi wllhoutside
the tension when
in cm H20 at 30 C thread cation stood
s ponge exceeded opened
Tom
1 69 Fine 2 min 06 sec Fine 1 cm Fine 3.5 kg through the 3cm
sponge
36
WO 20111058316 PCT/GB2010/002077
Same as
2 67 Fine 2 min 09 sec Fine 1.5 cm Fine 5.8 kg 3cm
above
Same as
3 66 Fine 3 min 53 sec Small 1.5 cm Fine 4.06 kg 3cm
above
Unable to
4 64 Fine 3 min 06 sec Fine 1.7 cm Fine 4.89 kg 3cm
break
Unable to
5 68 Fine 3 min Fine 1 .7 cm Fine 5.7 kg break or 3cm
separate
Unable to
6 68 Fine 2 min 09 sec Fine 1.6 cm Fine 5.67 kg 3cm
break
Tore
7 66 Fine 2 min 22 sec Small 2 cm Fine 4.35 kg through the 3cm
sponge
Same as
8 67 Fine 3 min 30 sec Fine 2 cm Fine 2.78 kg 3cm
above
Some as
9 66 Fine 3 min 02 sec Fine 2 cm Fine 5.9 kg 3cm
above
Unable to
break or
10 68 Fine 4 min 32 sec Fine 2 can Fine 5.49 kg separate 3cm
from the
sponge
In summer
5 Thus it can be seen that the old (known) device is inferior in several respects. The
Median
Time
length Loop Sponge
taken Pate when
of Capsule Inside Free Knot Weight size
to max
chord outside the end specific withsto when
open tension
in cm (Range) sponge of ation oil fully
in H2O exceeded
(Range (Range) threa open
at 30 C
of
6.02
0.8cm 2.41kg
Old 77 cm sec 2.8cm Thread
(0.710 Variable (1.42kg
Sponge (75- (4 to Fine Fine (2.5(o snapped in
0.9 knots 103.10
Kit 78.5cm) 6,29se 3cm) all 10 tests
cm) kg)
c)
3.01
Majority -
New 67cm min Two half 4.62kg
thread tore
Sponge (64 to (2.06 Fine Fine 1cm hitch (2.78 to 3cm
Through the
Kit 69cm) to 4.32 knot 5.8)
sponge
min)
advantageous performance of the device of the invention can be clearly
appreciated.
37
WO 2011/058316 PCT/GB2010/002077
In addition it should be noted that in in vivo tests, there have been no losses of the
device in subjects , as compared to the known old device which has regularly been lost
in subjects.
5
All publications mentioned in the above specification are herein incorporated by
reference. Various modifications and variations of the described aspects and
embodiments of the present invention will be apparent to those skilled in the art without
departing from the scope of the present invention . Although the present invention has
10 been described in connection with specific preferred embodiments , it should be
understood that the invention as claimed should not be unduly limited to such specific
embodiments. Indeed, various modifications of the described modes for carrying out
the invention which are apparent to those skilled in the art are intended to be within
the scope of the following claims.

Claims
1. A swallowable cell sampling device comprising an abrasive material capable of
5 collecting cells from the surface of the oesophagus, and a means for retrieval wherein
the means for retrieval comprises a cord, characterised in that the cord is attached to
the abrasive material by means of a hitch knot.
2. A device according to claim 1 wherein said hitch knot is a double overhand
10 knot
3. A device according to claim 1 or claim 2 wherein said abrasive material is
compressible.
)5 4. A device according to any preceding claim wherein said abrasive material
comprises reticulated polyurethane.
5. A device according to any preceding claim wherein said cord is attached to
said abrasive material via a loop of cord arranged below the surface of the abrasive
20 material, said loop being closed by the hitch knot.
6. A device according to any preceding claim wherein said abrasive material is
compressed and wherein said abrasive material is retained in a compressed state by a
soluble capsule.
25
7. A device according to claim 6 wherein said soluble capsule comprises a
gelatine capsule. %
8. A device according to claim 7 wherein said capsule Is capable of dissolution
30 and the compressible abrasive material is capable of reverting to its uncompressed size
within 5 minutes upon immersion in water at 30 degrees Celsius.
9. A device according to any preceding claim wherein the device comprises an
unswallowable element at the end distal from the swallowable abrasive material.
35
10. A kit comprising a device according to any preceding claim, and reagent for
use in the detection of a cellular marker.
39
WO 2011/058316 PCT/GB2010/002077
11. A method for aiding the diagnosis of Barrett's oesophagus or Barrett's
associated dysplasia in a subject, said method comprising sampling the cellular surface
of the oesophagus of said subject with a device according to any of claims I to 9 and
assaying the cells for a cellular marker, wherein detection of such a marker indicates
5 increased likelihood of the presence of Barrett's or Barrett's associated dysplasia.
12. A kit according to claim 10 or a method according to claim 11 wherein the
marker is selected from the group presented in Table I.
10 13. A kit according to claim 10 or a method according to claim I I wherein the
marker is selected from the group presented in Table 2.
14. A kit according to claim 10 or a method according to claim 11 wherein the
marker is selected from the group presented in Table 3.
15
15. A device, kit or method as disclosed herein with reference to one or more of the
accompanying drawings.