Note: Descriptions are shown in the official language in which they were submitted.
WO 95/14927 PCT/CA94100646
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SCREENING TEST FOR EARLY DETECTION OF
COLORECTAL NEOPLASIA
FIELD OF INVENTION
This invention relates to a simple screening test for
colorectal cancer. Specifically, a method is described
whereby a colorectal cancer marker is detected in rectal
mucus obtained by digital rectal examination. More
particularly, this marker is detected in the mucus deposited
on a support using Schiff's reagent.
BACKGROUND OF THE INVENTION
Colorectal carcinoma is the second most frequent cause
of cancer mortality in men and women, causing nearly one
third of all malignancy-related deaths in North America. It
has been estimated that ultimately as many as 6 % of Canadians
and Americans will develop malignancy in the lower bowel, and
over 50~ of them will die within 5 years of diagnosis.
Because there are no realistic prospects of significantly
improving the cure rate once the cancer has spread beyond the
bowel wall, many authorities believe that colorectal cancer
can be controlled only by preventive measures (1).
Primary prevention, i.e. averting the development of the
tumour by altering biological risk factors, is not yet
feasible since so little is understood of the etiology of the
disease. Alternatively, secondary preventive measures, i.e.
detection at an asymptomatic, treatable state, would be
possible should an effective screening test be available.
Indeed, neoplasms of the lower bowel have the characteristics
that make them a suitable candidate for the development of
a screening test. This is because i) it is a common cause
of cancer-related deaths, and ii) whereas once the stage of
true cancer is reached, leading to symptoms, the mortality
rate is over 50~, removal of bowel neoplasms at its earliest,
asymptomatic stage can be done by non-surgical endoscopic
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polypectomy, without any significant risk. Moreover, it
requires at least four to six years before an adenomatous ,
polyp reaches the cancer stage, so there is ample opportunity
to detect these neoplasms at their treatable stage. Recent
clinical studies document a decrease in mortality from
colorectal cancer screening, as predicted by these
theoretical considerations. The problem to-date has been
that polyps can be reliably detected only by endoscopy.
Thus, colorectal cancer satisfies each of the following
three criteria of a disease considered suitable for a
screening program. First, it is a relatively common
condition with serious consequences. Second, curative
treatment is available when detected at an early stage, i.e.
snare polypectomy through a colonoscope or surgical segmental
bowel resection. Third, the prevalence is sufficiently high
to justify the expense of a screening program (2).
PRINCIPLES OF SCREENING
The goal of a medical screening program is to reduce
morbidity and mortality by detecting a disease at a
sufficiently early stage to allow curative treatment. It is
not designed necessarily to diagnose a disease, but to
determine which asymptomatic, apparently disease-free
individuals should undergo diagnostic investigations. The
ability of a screening test to distinguish those who warrant
further evaluation from those who do not is expressed in
epidemiological terms. The term "sensitivity" is defined as
the proportion of diseased individuals who have a positive
test, i.e. the proportion of true positives/all persons with
the disease. °'Specificity" is the proportion of disease-free
subjects who have a negative test, i.e. the proportion of '
true negatives/all persons without the disease. The term
°'positive predictive value" is the proportion of positive '
tests due to the disease, i.e. the proportion of true
positives/all positives. Almost always, sensitivity and
specificity must be traded against each another.
Intuitively, it appears wise to design a screening test for
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a fatal disease so as to optimize sensitivity, in order to
detect as many individuals with the disease as possible.
It
has been emphasized, however, that optimizing sensitivity
brings with it a risk of reducing specificity to such an
extent that unacceptably high costs, poor compliance, and
"flooding" of diagnostic facilities result. Moreover,
positive predictive value, which is a particularly useful
expression of the value of a screening test, is critically
dependent on specificity and on the prevalence of the disease
in the population screened.
It has been stressed that the effectiveness of a
screening test can be properly evaluated only by randomized
controlled trials. In the case of cancer, it is not
sufficient to demonstrate that life is prolonged when the
malignancy is detected by a positive screening test, compared
to when the tumour is diagnosed after the development of
symptoms. Instead, it must be shown that screened
individuals have a lower death rate from the malignancy than
similar individuals not enrolled in such a screening program.
Important sources of error in interpreting the results of
previous screening programs include lead time bias, length
bias, and patient selection bias. A particularly fallacious
assumption is that the predictive value of a screening test
is the same in a hospitalized population with advanced
disease, in which the test is usually initially tried, as
it
is in a healthy popa.ilation with early minimal disease,
to
which the test is usually aimed.
CURRENT POPULATION SCREENING METHODS
Endoscopic methods, such as sigmoidoscapy or entire-
length colonoscopy, are diagnostic rather than screening
techniques. The only current method of colorectal cancer
screening in the general population is searching far occult
blood in the stool {3). Present techniques e.g. HemOccult*
II involve smearing a sample of stool onto' guaiac impregnated
paper which, after treatment with hydrogen peroxide
containing developer, exhibits blue colour if blood,
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haemoglobin, is present. After almost two decades of
experience with this methodology, it has become clear that, ,
even in expert centres, the sensitivity is less than 50% for'
curable neoplasms, and that the positive predictive value
4
approximates, at best, only-40% in a clinic population. An
update from the large-scale (n=97,205) University of
Minnesota, Minnesota, United States, prospective trial
indicates a positive predictive value for colorectal cancer
*
of only 2.2% when HemOccult is used in asymptomatic subjects,
aged 50-80, with an overall disease prevalence of 0.2% (4).
Furthermore, factors such as medications, multiple dietary
constituents, delays in specimen handling, variabilities in
fecal hydration, and storage of assay materials commonly
confound results. Analysis of one of the three randomized
controlled studies assessing the value of HemDccult* suggests
comparable mortality rates in the screened and control
populations (5). Newer methods of detecting occult blood,
e.g. methods based either on porphyrin analysis [HemoQuant~
or antibody specif is far human haemoglobin, may improve on
these results. However, three limiting problems remain
unlikely to be overcome. These are that colorectal
malignancies shed blood only intermittently, upper
gastrointestinal tract bleeding may make the results
(falsely) positive, and multiple lesions in the lower bowel,
apart from colorectal neoplasms, commonly bleed. such
lesions include :hemorrhoids, diverticulae, .ulcers, and
vascular ectasie. Compliance in unselected populations has
been estimated to be less than 30%, at least partly because v
the technique requires patients themselves to smear their
stool onto a slide or a strip, a task most people find not
only distasteful, but also technically difficult. Despite °
this, FIemOccult* continues to be widely used because the
American Cancer Society has recommended occult blood testing °
yearly for all individuals over 50 years of age, arguing that
even an imperfect test will save many lives. Implicit in all
arguments over the value of HemOccult*is that any improvement
in screening techniques for bowel malignancy would have a
* Trademark
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dramatic impact on colorectal cancer mortality rates from the
disease, since the screening for occult blood even in the
present form leads to reducing mortality from colorectal
cancer (6).
EXPERIMENTAL SCREENING METHODS
(i) Screening for colorectal cancer by stool DNA analysis
(7) .
This is based on the presence in stool of neoplastic
l0 cells shed in large numbers into the colonic lumen. In
principle, a mutation which is common to neoplasms could be
detected with high precision by analyzing DNA from these
cells. Therefore, the existence of a detectable mutation in
the colorectal tumour is a prerequisite for developing such
a method of screening. Unfortunately, this technique can
recognize a mutation based only on a new or altered
oligonucleotide sequence, but not on a loss of its portion.
Thus, neoplasia-related mutations based on deletion in genes,
e.g. allele losses on chromosomes such as are commonly found
in colorectal tumours, are beyond the limits of the
methodology. Currently, the most common mutation is the
K-ras oncogene mutation present, in about 40% of colorectal
carcinomas and adenomas. Screening for K-ras gene can
therefore detect, at best, only 40~ of all neoplasias. This
methodology is at present technically very complex and
expensive.
(ii) Screening for the presence in colonic mucin of a
cancer-related disaccharide, D-Galp(131-
3)-D-GalpNAc(al,Ser/Thr), T-(Thomsen-Friedenreich) antigen,
since it is widely known that T-antigen is not expressed by
cells in healthy colons, whereas it is expressed by cancer
(8) .
(a) Monoclonal antibodies and lectins: It has been shown that
monoclonal antibodies raised against synthetic T-antigen
recognize and bind to cancer cells. Similarly, peanut
agglutinin (PNA), a lectin, binds strongly to the same
disaccharide, but recognizes malignancy with lesser
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specificity. Amaranthin, a lectin from Amaranthus caudatus,
has been reported to have better specificity for T-antigen ,
than PNA. Neither amaranthin nor PNA bind to histological
sections of normal mucosa, but both bind to mucin in the
Y
goblet cells of tumours and certain polyps, and in the
transitional mucosa. The visualization of the binding
utilizes fluorescently labelled antibodies and lectins (9).
(b) Galactose oxidase test. T-antigen is also reported to
be detectable colorimetrically after oxidation of oH-6 of
galactose using galactose oxidase and visualization of the
resulting aldehyde with Schiff~s reagent, - United States
Patent No. 4857457, issued August 15,.1989 to Shamsuddin et
al. In contrast with the tests using lectins, this test is
performed on mucus samples obtained by digital rectal
examination and smeared onto a support. This system
demonstrated a sensitivity of 74% and specificity of 50% for
colorectal neoplasms, i.e. adenomatous polyps and cancer, in
ones study with only 1 false negative result among 59 patients
with cancer. Since then a number of reports of basically the
same test have appeared with sensitivity ranging from 35% to
100% and specificity ranging from 15% to 76%. Some
investigators found that the test was more sensitive, but
less specific, than HemOCCUlt*. The lesser specificity has
bean ascribed to the positivity of test in individuals with
certain inflammatory condition, such as diverticulitis and
ulcerative colitis (10).
,~t.~'ERENGE LI ST
The present specification refers to the following
publications.
PUBLICATIONS
I. Lieberman DA: Targeted colon cancer screening: A concept
whose time has almost come. Amer. J. Gastroenterol. 1992, ~7,
* ~s.~ra~emark
-WO 95114927 2 I ~ 6 5 ~ ~ pCT/CA9.1/006.16
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1085.
II. Eddy DM: Screening for colorectal cancer. Ann. Int. Med.
1990, 113, 373.
III. Rex DK, Lehman GA, Ulbright TM, Smith JJ, Pound DC,
Hawes RH, Helper DJ, Wiersema MJ, Langefeld CD, Li W: Colonic
neoplasia in asymptomatic persons with negative fecal occult
blood tests: influence of age, gender, and family history.
Am. J. Gastroenterol. 1993, 88, 825.
IV. Mandel JS, Bond JH, Bradley M, Snover DC, Church TR,
Williams S, Watt G, Schuman LM, Ederer F, Gilbertsen V:
Sensitivity, specificity, and positive predictivity of the
Hemoccult test in screening for colorectal cancer.
Gastroenterol. 1989, 97, 597.
V. Selby JV, Friedman GD, Quesenberry, Jr. CP, Weiss NS:
Effect of fecal occult blood testing on mortality from
colorectal cancer. Ann. Intern. Med. 1993, 118, 1.
VI. Mandel JS, Bond JH, Church TR, Snover DC, Bradley GM,
Schuman LM, Ederer F: Reducing mortality from colorectal
cancer by screening for fecal occult blood. New Engl. J. Med.
1993, 328, 1365.
VII. Editorial: Screening for colorectal cancer by stool DNA
analysis. Lancet 1992, 339, 1141.
VIII. Boland CR. Montgomery CK, Kim YS: Alterations in
human colonic mucin occurring with cellular differentiation
and malignant transformation. Proc. Natl. Acad. Sci. USA
1982, 79, 2051.
IX. Rinderle SJ, Goldstein IJ, Matta KL, Ratcliffe RM:
Isolation and characterization of Amaranthin, a lectin
present in the seeds of Amaranthus caudatus, that recognizes
the T- (or cryptic T) antigen. J. Biol. Chem. 1989, 264,
16123.
X. Sakamoto K, Muratani M, Ogawa T, Nagamachi Y: Evaluation
of a new test for colorectal neoplasms: a prospective study
of asymptomatic population. Cancer Biotherapy 1993, 8, 49.
XI. Robins JH, Abrams, GD, Pincock JA: The structure of
schiff reagent aldehyde adduct and the mechanism of the
Schiff reaction as determined by nuclear megnetic resonance
WO 95/14927 PCT/CA9.1/006.tG
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spectroscopy. Can. J. Chem. 1980, 58, 339.
XII. Kasten FH: The chemistry of Schiff's reagent. 1960, Int.
Revs. Cytol. 10, 1.
XIII. Shamsuddin A: Diagnostic assays for colon cancer.
CRC Press, Boca Raton FL, 1991.
PATENTS
1. U.S.P. 4857457, Shamsuddin et al, August 15, 1989.
2. U.S.P. 4762800, Rettig et al, August 9, 1988.
3. U.S.P. 4863854, Mattes et al, September 5, 1989.
4. U.S.P. 4962187, Pant, October 9, 1990.
5. U.S.P. 5073493, Yamashina, Dec ember 17, 1991.
6. U.S.P. 5008184, Linnane, April 16, 1991.
SUMMARY OF THE INVENTION
In contrast to the above-noted prior art, we have
surprisingly discovered that mucus collected from individuals
with neoplastic disease of the colorectum contains a marker
which produces coloration with Schiff's reagent. This assay
does not require detecting the disaccharide marker beta-D-
Gal(1->3)-D-GalNAc, as required by the prior art. In fact,
this disaccharide does not react with the Schiff's reagent.
It is an object of the present invention to provide a
tool for the screening of asymptomatic persons for cancer of
the large bowel and rectum.
It is a further object of the present invention to
provide a screening test to detect neoplasms of the large
bowel and rectum prior~to development of a bleeding cancer.
It is yet a further object of the present invention to
provide a screening test for colorectal cancer which does not
give a high percentage of false positive and false negative
results.
It is still yet a further object of the present
invention to provide a screening test kit by means of which
said test can be conducted outside of a hospital, medical
CA 02176508 2004-07-13
labotary or clinic.
These and other objects and advantages of the invention
will be seen from a reading of the specification as a whole.
Accordingly, the invention provides in one aspect a
method for detecting the presence of precancer or cancer of
the colon or rectum, which method comprises obtaining a sample
of colorectal mucus from the rectum of a patient; treating
said sample with Schiff's reagent and detecting precancer or
cancer of the colon or rectum based upon the coloration
produced in said sample by said treatment.
Thus, the invention is a method for detecting the
presence of neoplasia or cancer of the colon or rectum, which
method consists essentially of obtaining a sample of
colorectal mucus from the rectum of a patient; treating said
sample with Schiff's reagent; and detecting neoplasia or
cancer of the colon or rectum based upon the coloration
produced in said sample by said treatment.
For the purpose of clarification the process of the
invention does not require treatment of the mucus sample with
an enzyme for detecting the disaccharide marker beta-D-gal
(1>3)-D-GaLNAC prior to treatment with Schiff's reagent.
More particularly, the present invention relates to a
method for detecting the presence of neoplasia or cancer of
the colon or rectum in a patient, which method comprise
treating a sample of colorectal mucus with Schiff's reagent,
without a step of adding an enzyme for detecting the
disaccharide marker beta-D-gal (1-> 3)-D-GalNAc; and detecting
9
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neoplasia or cancer of the colon or rectum based upon the
coloration produced in the sample by the treatment.
The present invention also relates to a method for detecting
the presence of neoplasia or cancer of the colon or rectum in
a patient, which method consists essentially of treating a
sample of colorectal mucus with Schiff's reagent, without a
step of adding an enzyme for detecting the disaccharide marker
beta-D-gal (1-> 3)-D-GalNAc; and detecting neoplasia or cancer
of the colon or rectum based upon the coloration produced in
the sample by the treatment.
The aforesaid coloration does not develop with basic
fuchsin alone, although the Schiff's reagent itself is
prepared from basic fuchsin (11) .
The important advantage of testing rectal mucus, compared
to lectin or antibody binding to histological sections of
tumour tissue, is the easy accessibility of the material to be
tested. Since the luminal surface of the colon is lined
throughout its length with mucus, a viscoelastic gel composed
of water, electrolytes, organic chemical substances, and large
molecular weight glycoproteins (mucins), as well as sloughed
cells and bacteria, which is movable along the bowel, it is
suggested that rectal mucus contains mucus from the entire
colon, i.e., the mucus secreted by a neoplastic tissue flows
along the bowel into the rectum at which point it is sampled.
A mucus sample obtained by a physician or a trained
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nurse from a screened subject is deposited on a suitable
water-insoluble substrate or support, such as a pad or disc. .
Suitable support materials are, for example, glass
microfibres Whatman GF/C, polymer fibres such as Siotrace RP,
Metrical*BM 450, Metrical*VM-l, Sepraphor~ ITI, Versapore
450, or cellulose fibre such as Whatrnan* 3MM. The support may
or may not be pre-treated with an antioxidant such as BHT
(butylated hydroxytoluene) or BHA (butylated hydroxyanisol}.
Two procedures are preferably employed.
1o In procedure A, the mucus sample is deposited on a
support as described hereinbelow, the mucus-carrying support
is rinsed in potassium phosphate buffer, generally for about
minutes, then water, excess water removed and the support
placed in Schiff's reagent for a short period of time, such
as 1 minute, washed briefly with distilled water, and dried,
for example in air or by pressing it between two stacks of
filter paper or both. A positive reaction is snored When a
purple-magenta colour appears on the filter in 20-25 minutes.
In procedure B, the mucus sample is deposited on a
support already containing Schiff°s reagent, as described
hereinbelow. The support develops the purple-magenta colour
within a short period of time, such as 30-60 seconds after
the mucus specimen is deposited, if the marker is present.
The support, such as a pad, is, for procedure B, dipped
in or otherwise treated with the solution of Schiff's reagent
of appropriate strength to provide an effective, suitable
amount retained an the pad to effect appropriate detection
by colouration of the marker.
We have observed that the mucus smeared-pad, when
exposed to air for prolonged periods~of time, usually at
least one hour, after treatment with Schiff°s reagent,
becomes uniformly coloured, due to the oxidation of the
Schiff°s reagent. Although a true positivity is readily
distinguished from scuch background, such background can be
further minimized by antioxidant pretreatment as hereinbefore
described.
If a mucus specimen does not produce any coloration, it
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is either because of the absence of the marker in the mucus,
or because mucus was not collected by the gloved finger and
therefore not deposited on the support. To distinguish
between these two possibilities, the negative-testing mucus-
treated support is treated with 0.5% periodic acid solution
for 5 minutes, rinsed with water, stained with Schiff's
reagent and rinsed again. When mucus lacking the marker is
present, purple-magenta coloration appears; otherwise the
support remains colourless, although some background
coloration may develop.
During the practice of the process according to the
invention, different shades of colour are sometimes observed.
Such variations may reflect structural differences in the
marker and could possibly correlate with clinical condition
of the subject, e.g., chronic inflammation, ulcerative
colitis, and the like.
It is known that the chemical properties of Schiff's
reagent vary according to its method of preparation.
Accordingly, a number of these variants were prepared and
tested for use in the process of the present invention. The
particulars of the various methods of preparation are listed
hereinbelow. We have found that differences in colouration,
sensitivity, specificity and oxidisability were obtained.
While providing reasonably reproducible results, some of the
Schiff's reagents listed below, for instance, were either
overly sensitive, or poorly sensitive to the mucus specimen
or gave a less than satisfactory background colouration. To
obtain reproducible results with maximum sensitivity, the
preferred Schiff's reagent No. 1 described below has been
developed.
It is noted that commercial basic fuchsin (p-rosanilin)
is often quite impure and Schiff's reagent is preferably
prepared therefrom by purification before use. Sulphur
dioxide, necessary for the preparation of the reagent, can
be used as such in the gaseous form, or generated in situ
from various precursors, such as NaHS03, SOC12, KzS205 and
Na2S205, ( 12 ) . We have found that the method of preparation of
CA 02176508 2004-07-13
the Schiff's reagent determines to some degree the reactivity,
sensitivity, and stability of the reagent.
In a further aspect, the invention provides a screening
kit comprising a container such as a package, carton, tube,
box, roll, tape, or other capsule like object comprising a
water-insoluble substrate capable of absorbing colorectal
mucus and Schiff's reagent.
More particularly, the present invention provides to a
screening kit for detecting the presence of neoplasia or
cancer of the colon or rectum, which kit comprises a container
comprising a water-insoluble support for absorbing a
colorectal mucus and Schiff's reagent or a source of Schiff's
reagent, provided that the kit does not include an enzyme for
detecting the disaccharide marker beta-D-gal (1-> 3)-D-GalNAc.
Keeping the above in mind, the present invention also
relates a screening kit for detecting the presence of
neoplasia or cancer of the colon or rectum, which may kit
consist of a container comprising a water-insoluble support
for absorbing a colorectal mucus, and Schiff's reagent or a
source of Schiff's reagent.
Accordingly the present invention further provides a
screening kit for detecting the presence of neoplasia or
cancer of the colon or rectum, which kit consists of a
container comprising a water-insoluble support for absorbing a
colorectal mucus and a description of a method for detecting
the disaccharide marker beta-D-gal (1-> 3)-D-GalNAc from the
mucus by treating the colorectal mucus with Schiff's reagent,
without a step of adding an enzyme for detecting the
disaccharide marker beta-D-gal (1-> 3)-D-GalNAc; and detecting
neoplasia or cancer of the colon or rectum upon the coloration
produced in the sample by the treatment
The support (substrate) may have been pre-treated with a
solution of the Schiff's reagent to retain an active portion
thereof; or the container may have each of the support and the
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Schiff's reagent separately packaged; or the Schiff's reagent
may be regenerated before use from basic fuchsin deposited on
the support.
In a further aspect, the invention provides a screening
kit as a hereinabove defined, but incorporating packaged basic
fuchsin instead of Schiff's reagent by subsequent reaction of
sulphur dioxide with the basic fuchsin.
More particularly, the present invention provides a
screening kit for detecting the presence of neoplasia or
cancer of the colon or rectum, which kit may consist of a
container comprising a water-insoluble support for absorbing a
colorectal mucus and basic fuchsin for the subsequent
generation of Schiff's reagent.
DESCRIPTION OF PREFERRED EMBODIMENTS
In order that the invention may be better understood
preferred embodiments will now be described by way of example
only, with reference to the accompanying drawing wherein the
Figure shows a perspective view of an apparatus of use in the
practice of the invention.
The Figure shows generally as 10, a frame assembly
comprising a pair rectangularly-shaped plates 12 and 14 (lOcm
X lOcm X 3mm) formed of Plexiglass° thermoplastic. Upper
plate 12 is operably superimposed upon lower plate 14 in firm
abutment together by means of a pair of opposed clips 16.
Plate 12 has a pair of circular apertures 18 (2 cm
diameter). Retained between plates 12, 14 directly below
apertures 18, as to be accessible therethrough, is a pair of
disc supports 20, formed of Sepraphore* III polymer film
(2.5cm diameter) .
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In operation, a physician or nurse, for example, smears
a mucus specimen onto the surface of each of pads 20, via
apertures 18. Clips 1.6, are released and discs 2o processed
as hereinbelow described, either in reference to Procedure
A, or the subsequent results read directly in frame assembly
to according to procedure B. Discs 20 are subsequently
removed and discarded. Plates 12 and 14 are generally washed
and re-used.
Two general procedures are hereinbelow described as a
screening test for the early detection of colorectal
neoplasia.
Procedure A:
A Sepraphore III support in the form of a disc, upon
which a mucus specimen, obtained during the digital rectal
examination, is smeared, is fastened in a frame formed by two
square Plexiglass~ plates, which may be conveniently handled
in a physicians office. A suitable lubricant, such as
' glycerol, for the rectal examination is chosen from among
those that do not .react with Schiff's reagent. For
2U processing, the following method has been found to be
suitable, because it minimizes background coloration.
A support bearing a smeared-on mucus specimen is placed
in O.1M potassium phosphate buffer (pH 6.5-6.?) for 10
minutes, taken out and washed briefly with distilled water.
Excess water is removed by placing the support on a cellulose
filter with -the side bearing the mucus smear in an up
position. The support is then planed in Schiff's reagent
denoted No.l described hereinbelow, for 1 minute, taken out,
washed briefly with distilled water, squeezed between two
30. cellulose filters, and air dried. A positive reaction is
scored when a purple-magenta colour appears on the filter
within 20-25 minutes. Omitting the buffer wash leads to a
decreased test specificity even if the sensitivity is
maintained.
Stools deposited on the support together With the mucus
cause an unwanted transformation in deposited mucus to take
place during the storage before development, to result in a
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positive test reading. To prevent this transformation from
happening, a pretreatment of the mucus free support is
carried out before use with 0.1% solution of an antioxidant,
such as, for example, BHT in 95% ethanol, or BHA.
Two mucus samples are preferably obtained from each
subject, one sample is used for testing and the other used
for confirmatory purposes. It should be noted that usually
unequal quantities of mucus are found deposited on the
supports. The specimens are then treated with the Schiff's
l0 reagent, the results are recorded, and the specimens further
treated with periodic acid-Schiff's reagent to determine the
quantity of mucus deposited. This procedure has shown that
a weakly positive mucus-Schiff test result is to be expected
if only a small amount of mucus is present on the support.
This has, thus, the same validity as a strongly positive
result of an abundant mucus sample.
Procedure B: A suitable support is prepared as follows:
A cellulose pad or disc (Whatman*3N~I) is soaked in 0.1%
solution of BHT in 95% ethanol and dried. Then it is soaked
in Schiff's reagent r~l, and dried. This pre-treated support
can be stored at -20 °C, or sealed to prevent contact with
air. Alternatively, the support after deposition thereon of
Schiff's reagent, far additional protection, may be again
soaked in BHT solution and dried.
The specimen of mucus obtained on a gloved finger after
digital sampling is smeared on this support. The specimen
is positive for the marker if the coloration develops within
approximately up to one minute after the specimen was
deposited on the support. Later colour development does not
represent positivity.
Since the treatment with phosphate buffer solution is
omitted in procedure B, the specificity of procedure B is
lower than that of procedure A (from 80-90% to 60-70%).
Since the sensitivity remains close to 100%, the negative
results represent the disease-free individuals. Positive
results can be reexamined by procedure A to increase
specificity of the testing.
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A number of Schiff's
reagents of use in
the invention
are prepared as fol lows.
1. Basic fuchsin (0.2 g) is dissolved in hot water (100
mL), boiled for 5 minutes, filtered and cooled to room
temperature. Sodium
bisulfite (1.17 g)
and 1N hydrochloric
acid (17 mL) are ad ded sequentially to the filtrate and
the
solution allowed to stand in a dark place at room temp. for
4 days. Decolorizing
charcoal (0.15 g) is
added, mixed well,
and filtered off. The resulting colourless or slightly
yellow solution is stable for suitable prolonged periods
of
time. The reagent is stored at +4C in a refrigerator.
2. Basic fuchsin (1.0 g) is dissolved in hot water (200
mL), boiled for 5 minutes, filtered and cooled to room
temperature. Then sodium
metabisulfite (1.0
g) and 1N
hydrochloric acid (20 mL) are added sequentially to the
filtrate and the solution
allowed to stand in
a dark place
at room temp. for 4 days. Charcoal (0.3 g) is added, mixed
well, and filtered off.
3. Basic fuchsin (1.0 g) is dissolved in hot water (200
mL), boiled for 5 minutes, filtered and cooled to room
temperature. Then potassium metabisulfite (1.0 g) and 1N
hydrochloric acid (20 mL) are added sequentially to the
filtrate and the solution
allowed to stand in
a dark place
at room temp. for 4 days. Charcoal (0.3 g) is added, mixed
well, and filtered off.
4. Basic fuchsin (1.0 g) is dissolved in hot water (200
mL), boiled for 5 minutes, filtered and cooled to room
temperature. Then potassium metabisulfite (1.0 g) and 1N
hydrochloric acid (25 mL) are added sequentially to the
filtrate and the solution
allowed to stand in
a dark place
at room temp. overn ight. If the solution is still coloured,
two drops of 6N hydrochloric
acid are added, stored
in a dark
place for 48 hours. Then charcoal is added, mixed well, and
filtered off.
5. Basic fuchsin (0.2 g) is dissolved in hot water (100
mL), boiled for 5 minutes, filtered and cooled to room
temperature. After thionyl.chloride (7-8 mL) is added to
the
WO 95114927 : ., ~ PCT/CA94/0064G
- 16 -
filtrate, the solution is allowed to stand in a dark place
at room temp. for 24 hours. Charcoal is added to decolorize
the solution, mixed well, and filtered off.
6. Basic fuchsin (1.0 g) is dissolved in 0.25M hydrochloric ,
acid (100 mL) and sodium bisulfite (10.0 g) is added. The
solution has pH=3.
7. Basic fuchsin (0.12 g) is dissolved in hot water (200
mL), boiled for 5 minutes, filtered and cooled to room
temperature. Then glacial acetic acid (11.40 mL) and sodium
bisulfite (1.0 g) are added sequentially to the filtrate.
The solution is slightly pink in colour.
8. Commercial Schiff's reagent (Fischer) pH=1 is adjusted
to pH=3 by the addition of aqueous sodium hydroxide (0.2N).
The resultant pink solution is decolorized with sodium
bisulfite.
9. Basic fuchsin (1.0 g) is dissolved in hot water (200
mL), boiled for 5 minutes, filtered and cooled to room
temperature. Then sodium bisulfite (1.0 g) and 1N
hydrochloric acid (25 mL) are added sequentially to the
filtrate and the solution allowed to stand in a dark place
at room temp. for 48 hours. Charcoal (0.3 g) is added, mixed
well, and filtered off.
10. Basic fuchsin (1.0 g) is dissolved in hot water (200
mL), boiled for 5 minutes, filtered and cooled to room
temperature. Then sodium bisulfite (2.0 g) and 1N
hydrochloric acid (25 mL) are added sequentially to the
filtrate and the solution allowed to stand in a dark place
at room temp. for 48 hours. Charcoal (0.3 g) is added, mixed
well, and filtered off.
11. To commercial Schiff's reagents (Sigma) pH=1.5 (100 mL)
is added glacial acetic acid (4.5 mL).
EXAMPLES '
Example 1
In all investigated groups, using modified procedure A,
clinically diagnosed cancers were detected with high
'WO 95114927 _ PCT/CA9410064G
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sensitivity, minimum 92%. In one study, out of 25 cancer
patients, 23 gave a positive result in the mucus-Schiff test;
of 30 large adenomatous polyps, 24 gave positives; of 76
small polyps, 43 gave positives.
It has been shown by the results in this group that
colonic inflammatory conditions such as ulcerative colitis,
diverticulitis, Crohn's disease, acute and chronic
inflammation often give positive results (Table). Since at
least some of these conditions have been recognized as risk
factors for colorectal cancer, these results show that the
test recognizes either susceptible individuals or an early
stage of neoplastic development. The latter argument applies
to polyps as well.
Example 2
A study was designed to compare patients visiting the
endoscopy unit with patients visiting physicians'
(gastroenterologists') offices for non-malignant disease. The
visits were due to unspecified complaints. The latter group
consisted of 45 patients and the former of 39 patients. Two
specimens were prepared from each patient. Modified
procedure A was used. The two main differences between the
groups were: (a) the endoscopy suite group received colonic
lavage beforehand to free the colon of faeces, and was on a
liquid diet for 24 hours prior to the mucus collection (and
colonoscopy), while (b) the other group did not receive
lavage and had no diet restrictions.
The results show positivity of 270 (95~ confidence
interval: 15.4-42.6), 12/45 among patients in physicians'
offices and 33~ (95~ confidence interval: 19.3-48.4), 13/39
among patients in the endoscopy suite. This study thus shows
that the presence of faeces in specimens, and normal food
intake, do not determine the percentage of the positivity.
The positivity is consequently due to a condition other than
colorectal cancer.
WO 9511-X927 PCT/CA9-t/OOG.t6
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Example 3
The specificity measured in clinical control populations
is very imprecise in patients, who at the moment of the test,
have no clinically detected neoplasms but have some other
unspecified ailments, which may well predispose to cancer
formation in the future. Thus, a study was designed to
determine the number of positives in a cohort of 47 young
adult volunteers unlikely to suffer of any intestinal ailment
or neoplastic condition. All individuals were between 18 to
35 years of age and feeling completely healthy. This
investigation was expected to provide an estimate of
specificity of the investigated screening tool. Four
specimens were collected from each individual.
The results described in Examples 2 and 3 suggest that
some individuals may have a condition increasing the risk of
neoplasia. The false positive rate among healthy young
volunteers (not patients) was found to be low (10.6%). A
segment of inflamed bowel may, in fact, be transformed into
a preneoplastic condition, and this perhaps is detected by
the test.
The high sensitivity and relatively high specificity of
the test for neoplasms may reduce the number of patients
undergoing colonoscopy because they have rectal bleeding,
unexplained iron-deficiency anemia, or a first-degree
relative with a tumour.
The Table shows the results of Schiff's reagent
analysis of mucus from a group of patients with
colorectal cancer and putative precancerous conditions at
the endoscopy unit at the Wellesley Hospital, Toronto,
Ontario, Canada, who agreed to submit themselves to the
mucus testing.
The following notes provide a better understanding
of the Table: .
(a) The two cancer patients listed as negative had
an ambiguous reaction due to very small amount
of mucus deposited on pads. They underwent
~WO 95114927 PCT/CA9-t/006.1G
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surgery before the test could be repeated;
(b) The positivity/negativity of polyps reflects
the well-known observation that some polyps
are cancer precursors whereas some are not;
(c) Laser treatment probably inhibits mucus
production by tumour, especially if the laser
coagulation is extensive;
(d) Positivity/negativity of the test in
previously removed carcinomas may reflect the
completeness of the cancer removal;
(e) Inflammatory conditions are considered a risk
factor for colonic cancer. The positivity in
the test may reflect how far an inflammation
has progressed to an early stage of cancer
development;
(f) *Percentage of groups with less than to
subjects are not calculated; and
(g) **CI, Confidence Interval of percentage
positive.
Although this disclosure has described and
illustrated certain preferred embodiments of the
invention, it is to be understood that the invention is
not restricted to those particular embodiments. Rather,
the invention includes all embodiments which are
functional or mechanical equivalents of the specific
embodiments and features that have been described and
illustrated.
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- 20 - '
TABLE
Diagnosis total # positive negative
of case # ~ 95~CI** #
Cancer 25 23 92 72.5-98.6 2 8
Polyp adenomatous76 43 57 45.1-68.1 33 43
( < 1 cm in diameter)
Polyp adenomatous5 3 2
( < 1 cm in diameter;
removed endoscopically
2-8 weeks prior
to
mucus collection)
Polyp adenomatous30 24 80 59.7-91.6 6 20
( < 2 cm in diameter)
Carcinoma (laser 6 1 5
treated)
Carcinoma (removed7 5 2
2-8 weeks before
mucus collection)
Ulcerative colitis6 4 2
Crohn's disease 7 3 4
Diverticulitis 1 1
Acute inflammation2 2
Chronic inflammation1 1
Acute colitis 1 1
TOTAL 167
