Note: Descriptions are shown in the official language in which they were submitted.
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SCREENING TEST FOR EARLY DETECTION OF
COLORECTAL CANCER
FIELD OF THE INVENTION
This invention relates to a simple screening test for colorectal cancer
whereby a
marker is detected in rectal mucus. 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. Many authorities believe that colorectal cancer
can be
controlled only by preventive measures (1) because there are no realistic
prospects of
significantly improving the cure rate once the cancer has spread beyond the
bowel wall.
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
suitable candidates for the development of a screening test. This is because
(i) they are a
common cause of cancer-related deaths, and (ii) whereas once the stage of true
cancer is
reached, and showing symptoms, the mortality rate is over 50%. Removal of
bowel
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neoplasms at their earliest, asymptomatic stage can be done by non-surgical
endoscopic
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 in consequence of 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 Screenina
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 interventions.
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/relative to 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/relative to 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/relative to all positives. Almost always, sensitivity and
specificity must be
traded against each another. Intuitively, it appears wise to design a
screening test for a
fatal disease so as to optimize sensitivity, in order to detect as many
individuals with the
disease as possible. It is emphasized, however, that optimizing sensitivity
brings with it
a risk of reducing specificity to such an extent that unacceptably high costs,
poor
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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.
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 population with early minimal disease,
to which the test
is usually aimed.
Current Population Screening Methods
Endoscopic methods, such as sigmoidoscopy or entire-length colonoscopy, are
diagnostic rather than screening techniques, although sigmoidoscopy is
sometimes used
for screening. The only current method of colorectal cancer screening in the
general
population is searching for occult blood in the stool (3). Present techniques
e.g.
HemOccult II which involves smearing a sample of stool onto guaiac-impregnated
paper
which, after treatment with hydrogen peroxide containing developer, exhibits
blue colour
if blood (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
approximates, at best,
only 40% in a clinic population. An update from the large-scale (n=97, 205)
University
of Minriesota, Minnesota, United States, prospective trial indicates a
positive predictive
value for colorectal cancer of only 2.2% (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 HemOccult suggests
comparable
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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
specific for human haemoglobin, 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 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, HemOccult 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 dramatic impact on colorectal cancer
mortality rates,
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 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. 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 complex
and expensive.
(ii) Screening for the presence in colonic mucin of a cancer-related
disaccharide, D-
Galp(ol-3)-D-GalpNAc(a1,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).
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(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 specificity. Amaranthin, a lectin from
5 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 goblet cells of tumours and certain polyps, and
in the
transitional mucosa. The visualization of the binding utilizes fluroescently
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, - U.S. Pat. No. 4,857,457, issued
Aug. 15,
1989; U.S.P. 5,348,860, issued September 20, 1994; and U.S.P. 5,162,202,
issued
November 10, 1992, 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
one study with only 1 false negative result among 59 patients with cancer.
Subsequently, a number of reports of basically the same test has 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 been ascribed to the positivity of the
test in
individuals with certain inflammatory conditions, such as diverticulitis and
ulcerative
colitis (10).
In contrast to the aforesaid prior art, a colorectal mucus assay not requiring
the
detection of the disaccharide marker beta-D-Gal(1->3)-D-GaINAc and a
saccharide
marker containing D-galactose and/or 2-acetamido-2-deoxy-D-galactose has been
described in U.S. Patent No. 5,416,025 to Krepinsky et al., issued May 16,
1995. In this
method, a sample of colorectal mucus is treated with Schiff's reagent, without
a step of
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6
adding an enzyme for detecting the aforesaid disaccharide marker, and
detecting the
color change in the sample.
The method described in U.S. Pat. No. 5,416,025 demonstrated a sensitivity of
92% for
colorectal cancer from a test with 25 cancer patients. However, specificity is
somewhat
compromised in that varying shades of pink coloration are often obtained and
result in
some false positives.
Although the screening test disclosed in U.S. Pat. No. 5,416,025 provides a
significant
improvement over aforesaid prior art methods in not requiring an enzyme pre-
treatment
step, and a reduction in the relative numbers of false positives and false
negative
results, it is still desirable to provide a simple assay which further reduces
the likelihood
of false positive and false negative readings.
REFERENCE LIST
The present specification refers to the following publications,
Publications
1. Lieberman D. A.: Targeted colon cancer screening: A concept whose time has
almost come. Amer. J. Gastroenterol. 1992, 87, 1085.
2. Eddy D. M.: Screening for colorectal cancer. Ann. Int. Med. 1990, 113, 373.
3. Rex D. K., Lehman G. A., Ulbright T. M., Smith J. J., Pound D. C., Hawes R.
H.,
Helper D. J., Wiersema M. J., Langefeld C. D., Li W.: Colonic neoplasia in
asymptomatic persons with negative fecal occult blood tests: influence of age,
gender,
and family history. Amer. J. Gastroenterol. 1993, 88, 825.
4. Mandel J. S., Bond J. H., Bradley M., Snover D. C., Church T. R., Williams
S., Watt
G., Schuman L. M., Ederer F., Gilbertsen V.: Sensitivity, specificity, and
positive
predictivity of the Hemoccult test in screening for colorectal cancer.
Gastroenterol.
1989, 97, 597.
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5. Selby J. V., Friedman G. D., Quesenberry Jr. C. P., Weiss N. S.: Effect of
fecal
occult blood testing on mortality from colorectal cancer. Ann. Intern. Med.
1993,
118,1.
6. Mandel J. S., Bond J. H., Church T. R., Snover D. C., Bradley G. M.,
Schuman L.
M., Ederer F.: Reducing mortality from colorectal cancer by screening for
fecal
occult blood. New Engl. J. Med. 1993, 328, 1365.
7. Editorial: Screening for colorectal cancer by stool DNA analysis. Lancet
1992,
339, 1141.
8. Boland C. R,. Montgomery C. K., Kim Y. S.: Alterations in human colonic
mucin
occurring with cellular differentiation and malignant transformation. Proc.
Natl.
Acad. Sci. USA 1982, 79, 2051.
9. Rinderle S. J., Goldstein I. J., Matta K. L., Ratcliffe R. M.: 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.
10. 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.
11. Robins J. H., Abrams, G. D., Pincock J. A.: The structure of Schiff
reagent
aldehyde adduct and the mechanism of the Schiff reaction as determined by
nuclear magnetic resonance spectroscopy. Can. J. Chem. 1980, 58, 339.
12. Kasten F. H.: The chemistry of Schiff's reagent. Int. Revs. Cytol. 1960,
10, 1.
13. Shamsuddin A.: Diagnostic assays for colon cancer. CRC Press, Boca Raton,
Fl.
1991.
Patents
U.S. Pat. No. 4,857,457, Shamsuddin et al Aug. 15, 1989
U.S. Pat. No. 4,762,800, Rettig et al. Aug. 9, 1988
U.S. Pat. No. 4,863,854, Mattes et al. Sept. 5, 1989
U.S. Pat. No. 4,962,187, Pant, Oct. 9, 1990
U.S. Pat. No. 5,073,493, Yamashina, Dec. 17, 1991
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U.S. Pat. No. 5,008,184, Linnane, Apr. 16, 1991.
U.S. Pat. No. 5,162,202, Shamsuddin, November 10, 1992
U.S. Pat. No. 5,348,860, Shamsuddin, September 20, 1994
U.S. Pat. No. 5,416,025, Krepinsky et al., May 16, 1995.
SUMMARY OF TBE INVENTION
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 an improved
screening test
to detect neoplasms of the large bowel and rectum prior to development of a
bleeding
cancer.
It is a yet further object of the present invention to provide a screening
test for
colorectal cancer which provides improved specificity.
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 neoplasia, precancerous condition or cancer of the colon or rectum
condition
thereof, which method comprises obtaining a sample of colorectal mucus from
the rectum
of a patient and detecting the presence of a marker selected from the group of
long chain
aliphatic aldehydes containing 12 - 20 carbon atoms, optionally, containing
olefinic
groups; most particularly C16 - C18 containing aliphatic aldehydes; and
plasmalogen-
bound precursors thereof.
More specifically, the invention provides a method for detecting the presence
of
neoplasia, a precancerous condition or cancer of the large intestine, which
comprises:
(a) obtaining a sample of large intestinal mucus from the rectum of a patient;
(b) assaying said sample to detect an aldehyde marker selected from the group
consisting of CH3(CH2)14CHO, CH3(CH2)16CHO, and precursors thereof,
and
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(c) detecting neoplasia, precancer or cancer of the large intestine based upon
the presence of the aldehyde detected in the mucus.
The marker is, preferably, detected immunochemically, and, optionally,
quantitatively. The precursors of the markers are believed to be plasmalogen-
bound.
The marker is preferably selected from the group consisting octadecanal,
common
name stearaldehyde, of the formula CH3(CH2)16CHO, hexadecanal, common name
palmitaldehyde, of the formula CH3(CHZ)14CH0, and 9-octadecenal, common name
olealdehyde of the formula CH3(CH2)7CH=CH(CH2)7CHO.
In preferred embodiments of the invention, the knowledge of the structures of
the
aldehydic markers enables observations of the presence of the aforesaid
aldehydes in
colorectal mucus utilizing specific properties of the aldehyde group, for
example, by
polarography or using reagents that specifically react with aldehydic group-
forming
compounds detectable by their resultant suitable properties, such as color,
for example,
specific spectral properties, fluorescence, mass spectral, chemi luminescence
and other
biological reactions detectable by color; and chromatographic properties.
It is believed that the aldehydes are released from acid-sensitive
plasmalogens
under acidic conditions, and, after their release, immediately react with the
reagent. The
excess of the unreacted reagent is most preferably removed, for instance by
repeated
washings with water and/or buffers. Many known aldehyde-detecting compounds
and
compositions may be of use in the practice of the invention. In particular,
compounds
containing amino groups that under acidic conditions form with aldehydes,
addition
compounds endowed with easily detectable properties, such as fluorescence or
color.
Examples, of such amino group-containing compounds are found in the group of
aniline-
based dyes. p-Rosanilin is a particularly suitable dye, since after being
transformed by
reaction with a sulfite or analogues in aqueous hydrochloric acid into
colorless Schiff's
reagent, the latter reagent reacts with aldehydes with high sensitivity to
form a purple
colored addition compound defined by absorbance at about X,,,,a,; 560 - 590
nm. The
utilization of p-rosanilin in the form of Schiff's reagent for detection of
aldehydes.in
colorectal mucus is described in more detail, hereinbelow.
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Preferably, the method comprises treating said sample with Schiff's reagent
and
detecting neoplasia or cancer of the colon or rectum based upon the coloration
produced
at about 560 - 590 nm Xn.,X in said sample by said treatment.
The specific coloration produced according to the practise of the present
invention
5 can be visually seen or detected by spectrophotometric determination at
about 560 nm.
Most preferably, the method does not require the additional step of enzyme
treatment for
detecting the disaccharide marker beta-D-Gal(1-3)-D-Ga1Nac(oc1-Thr/Ser) and a
saccharide marker containing D-galactose and/or 2-acetamido-2-deoxy-D-
galactose.
The present invention is based on the discovery that a narrower range of
colors
10 obtained by the action of Schiff's reagent on the components of mucus
collected from
individuals with neoplastic disease of the colorectum can be visually seen or
spectrophotometrically measured to better indicate true positives and reject
false
positives. We have discovered that the mucus collected from such individuals
contains
detectable amounts of the long chain fatty aliphatic aldehydes CH3(CH2)14CHO
and
CH3(CH2)16CH0, and the olefinic aldehyde CH3(CH2)7CH=CH(CH2)-7CHO, ~er se, or
are
bound within plasmalogens present in the mucus and released therefrom by the
acid of
the Schiff's reagent. We have found that a purple coloration having a light
absorption at
about 560 - 590 nm is produced by the Schiff s reagent with the aforesaid
aldehydes.
We have found that the mucus of neoplastic disease-free individuals does not
provide the
visually identifiable color spectrum seen in the mucus, from -individuals with
neoplastic
disease. Further, we have discovered that colorectal mucus contains basic
compounds
which cause the Schiff"s reagent to revert to the originating dye, p-
rosanilin; and produce
a pink-reddish coloration having a light absorption at 538 nm ~,.. It is often
difficult to
distinguish this non-desirable coloration from the sought for weak purple
coloration,
without the need of a color chart, and this, consequently, may lead to an
increase in false
positives and, thus, reduced specificity of the assay.
We have further discovered that although a number of low molecular weight
aliphatic aldehydes are present in colorectal mucus, these aldehydes, some of
which
contain a carboxylic acid function, by virtue of their solubility in water do
not interfere
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with the assay of the present invention, if, preferably, sufficient aqueous
washing of the
mucus is carried out.
Thus; in contrast to the above-noted prior art, if water-washing and strict
observation of the purple color at about 560 - 590 nm as the proper indicator
of the
presence of the high molecular aldehydes and, thus, the presence of a
precancerous
condition or cancer is maintained, a substantial reduction of false positives,
and
consequently a substantial increase in the specificity of the test, is
obtained.
The purple coloration produced according to the practice of the invention due
to
the presence of the aforesaid long chain aliphatic fatty aldehydes is
distinguishable from
the various shades of pink and red coloration caused by other substances
present in
colorectal mucus remaining in the mucus after aqueous water washing. A color
chart
enclosed to each kit assists in proper identification of the purple color,
even by untrained
persons, and thus enables an operator to maintain the high specificity of the
test.
It should be noted that the specific purple coloration of value in the
practice of the
invention does not develop with p-rosanilin alone, although the Schiff's
Reagent ~er se is
prepared from p-rosanilin.
We have found that the exact position of a,,,,,,X for a Schiff's Reagent-long
chain
aldehyde adduct is solvent dependent.
We have found that stearaldehyde - Schiffs Reagent adduct in dichloromethane
exhibits a relatively broad absorption maximum at about 590 1 nm, with a
shoulder at
about 555 1 nm. This adduct in ethanol shows an absorption at about 547 nm
with a
shoulder at about 578 nm. In the latter solution, the color changed from
purple to red
which we believe is due to instability of the adduct in ethanol. It is
insoluble in water.
P-rosanilin in water has an absorption at about 538 nm, but is insoluble in
dichloromethane.
Aldehydes of longer carbon chain length with Schiff's Reagent behave similarly
as stearaldehyde adduct. For example, the adduct of myristaldehyde in
dichloromethane
shows a maximum absorption at about 586 nm with a shoulder at about 556 nm.
Formaldehyde adduct in water gives a broad flat maximum extending from about
560 - about 593 nm.
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The important advantage of testing rectal mucus, compared to lectin or
antibody
binding to histological sections of tumor 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,
such as nucleosides and nucleotides, aminoacids, peptides, lipids including
phospholipids
and products of lipid oxidation, and large molecular weight glycoproteins
(mucins), as
well as sloughed cells and bacteria, which are movable along the bowel, it is
believed that
rectal mucus contains mucus from the entire colon, i.e., the mucus secreted by
a distal
neoplastic tissue flows along the bowel into the rectum at which point it is
sampled.
A general procedure of use in the implementation of the invention is as
follows.
A mucus sample obtained by a physician or a trained nurse using a gloved
finger
lubricated with MUKO or a similar lubricant which does not trigger any color
change in
Schifff's reagent during digital rectal examination from a screened individual
is deposited
on a suitable water-insoluble substrate or support, such as a pad or a disc.
Suitable
support materials are prepared from, for example, glass microfibres, some
polymer fibres
such as polyester fibres and cellulose or modified cellulose fibers. The
support may or
may not be pretreated with antioxidants such as BHT (butylated hydroxytoluene)
or BHA
(butylated hydroxyanisol).
The following procedure is preferably employed.
The mucus sample is deposited on a support as described hereinbelow, retained
thereon for about 90 minutes before rinsing, or, if taken from a freezer
allowed about 90
minutes to thaw. Subsequently, the mucus carrying support is rinsed in 0.1M
potassium
phosphate buffer, generally for about 10 minutes, twice washed with water for
2 minutes,
air dried for 15 minutes to remove the excess water, and the support placed in
Schiff's
reagent for a short period of time, such as 2 minutes, washed briefly with
distilled water,
and dried in air. A positive reaction is scored when a purple color appears on
the filter
within 20 - 25 minutes after removal from Schiff's reagent.
If a specimen does not produce any coloration, it is either because of the
absence
of the long chain aliphatic aldehydes or plasmalogen precursors in the mucus,
or because
mucus was not collected by the gloved finger and, therefore, not deposited on
the support.
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To distinguish between these two possibilities, a negative-testing support is
treated with
0.5% periodic acid solution for 5 minutes, rinsed with water, stained with
Schiff's reagent
for 5 minutes and rinsed again. When mucus lacking the marker is present,
purple
coloration appears at the place where the mucus was deposited; otherwise the
support
remains colourless, although some background coloration may develop.
During the practice of the process according to the invention, other colors
than
purple may be observed, in particular those leaning towards pink and red
tones. Such
colors reflect the presence of basic substances liberating the original pink-
red colored dye
p-rosanilin from colorless Schiff's reagent. Since the basic substances noted
hereinabove
are normally water-soluble, these variations usually indicate that the washing
of samples
prior to the treatment with Schiff's reagent was incomplete.
It is known that the properties of Schiff's reagent vary according to the
combination of various isomers present in commercial preparations of p-
rosanilin and
according to the method of preparation of Schiff's reagent itself. However, in
contrast to
aforesaid prior art, U.S. Patent No. 5,416,025 to Krepinsky et al., these
variations do not
exert a significant influence on the test because of the knowledge of the
aldehydic nature
of the marker and the chemical properties of the adduct between the aldehydes
and
Schiff's reagent prepared using a suitable procedure. The preferable procedure
for the
preparation of an appropriate Schiff's Reagent is described hereinbelow. To
obtain
reproducible results with maximum sensitivity and stability, it is desirable
to allow the
reagent to mature for between 4 days to 6 weeks in a refrigerator, i.e. at +3
-+5 C,
before use.
In a further aspect, the invention provides a screening kit comprising, for
example, a container such as a package, carton, tube, box, roll, tape or other
capsule-like
object comprising a water insoluble substrate capable of adsorbing colorectal
mucus and
wettable by water and aqueous solutions and by Schiff's reagent.
The substrate may generally be exposed through a suitable circular aperture
of,
say, for example, 1.0 - 1.3 cm diameter between two tightly sealed,
rectangular, hard
plastic plates using double-sided tape. The dimensions of the sealed assembled
plates
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may be those of microscope slides which would enable the utilization of the
equipment
standard for simultaneous development of microscope slides.
In operation, a physician or a nurse, for example, smears a mucus specimen
onto
the surface of the support in the plate. The plates are transferred to a
laboratory, where
they are processed in batches the size of which is determined by the equipment
utilized in
the practice of the test, for example, of ten plates, as hereinbelow
described. The plates
are discarded after the results are read.
A procedure is hereinbelow described as a screening test for the early
detection of
neoplasia of the large bowel and the rectum.
On to the support secured in the plates, as described hereinabove and
convenient
to handle in a physician's office, is smeared a specimen of mucus obtained
during the
rectal examination. A suitable lubricant, such as MUKO, for the rectal
examination is
chosen from among those that do not react with Schiff s reagent. For
processing, the
following method has been found to be suitable. Individual plates bearing
smeared-on
mucus specimen are placed into a holder carrying ten plates. The holder is
immersed into
a vessel containing 0.IM potassium phosphate buffer (pH 7.0) for 10 minutes,
while the
tank is gently, mechanically vibrated. When vibration is stopped, the holder
is lifted from
the tank, and the holder is subsequently immersed into a tank containing
distilled water
and gently vibrated for another two minutes. The water washing is repeated
once, the
holder is then lifted above the tank, and the excess water allowed to drip
back into the
tank for ten minutes. The holder with the plates is subsequently immersed into
another
tank containing Schi#f's reagent described hereinbelow, vibrated gently for 2
minutes,
then taken out and washed 3 times with distilled water by immersing it in a
water-
containing tank for 2 minutes in each case. The holder with the plates is then
air dried
and scored when purple color appears on the support within 20 - 25 minutes.
The
minutes are counted from the time of removal from Schiff's reagent. The color
is
compared with the color chart, and colors other than purple are counted as
negative.
Stools deposited on the support together with the mucus may cause an unwanted
transformation in the presence of air in the deposited mucus to take place
during storage
before development, which may result in a false positive test reading. To
prevent this
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transformation from happening, a pretreatment of the mucus-free support may be
carried
out prior to deposition of mucus with 0.1% solution of an antioxidant, such
as, for
example, BHT in 95% ethanol., or BHA.
If the test is negative by reason of no color on the support, it is useful to
establish
5 if mucus were deposited in the plate. To achieve this objective, the
specimen is then
treated with periodic acid-Schiff's reagent to determine whether the mucus was
deposited
on the plate. If the mucus is present, purple color appears. The smear often
shows
slightly yellow color when mucus is present; colorless deposit usually
indicates that only
colorless lubricant was deposited.
10 It should be noted that a weakly positive test result is to be expected if
only a
small amount of mucus is present on the support, and, thus, it has the same
validity as a
strongly positive result of an abundant mucus sample.
EXAMPLES
The results obtained, to-date, indicate that some individuals may have
presymptomatic malignancy, or a condition increasing the risk of neoplasia.
For instance,
a segment of inflamed bowel may be transformed into a preneoplastic condition,
and this
perhaps is detected by the test.
The high sensitivity 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.
Example 1
Preparation of the Schiff's reagent
Distilled water (220 mL) is brought to boiling, removed from heat source and p-
rosanilin (0.4 g) added. The mixture is stirred well and boiled again for 5
minutes, cooled
to 50 C, and the solution filtered through a folded paper filter. 1N
hydrochloric acid (34
mL) is added to the filtrate under stirring, and allowed to cool to room
temperature.
Sodium bisulfite (2.34 g) is added, stirred well and stored at room
temperature in a dark
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place for 4 days. A slightly straw-colored solution is obtained, to which
charcoal
(NORIT, 300 mg) is added and the mixture vigorously stirred for 1.5 minutes.
Subsequently, the solution is filtered through a double paper filter into a
dark glass bottle
and stored refrigerated at 3 - 5 C.
Example 2
Patients with colorectal cancer and putative precancerous condition.
The sensitivity of the test has been consistently very high since rarely a
cancer
was missed. However, the specificity measured in the clinical control
population is
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 development in the future. Previously, it was shown that the false
positive rate
among healthy young volunteers - not patients - did not exceed 10.6%.
Inflammatory
conditions of the large bowel are considered to increase cancer risk. A
segment of
inflamed bowel may, in fact, be transformed into a preneoplastic condition,
and this is,
perhaps, detected by the test. Table 1 shows the results of the test performed
on mucus
from a group of patients from the endoscopy unit of the Wellesley Hospital,
some of
these patients were diagnosed with colorectal cancer and putative precancerous
condition.
Table 1
This table shows the results from the endoscopy unit at Wellesley Hospital,
Toronto,
Ontario, Canada, who agreed to submit themselves to the mucus testing.
Positive Negative
Condition Total % 95% CI* % 95%CI*
Diverticular disease 4 3 1
Adenomatous polyp 3 3
Polyp <1 cm + 21 10 48 26-70 11 52 29-74
Polyp large >1 cm ++ 1 1
No Neoplasia 45 13 29 16-44 32 71 56-84
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Positive Ne agtiye
Condition Total # % 95% CI* % 95%CI*
Cancer 5 5
Follow up of
cancer removal+++ 5 3 2
Crohn's disease 2 1 1
Ulcerative colitis 4 3 1
*CI: confidence interval of percentage positive or percentage negative.
+Less than i cm in diameter
++More than 1 cm in diameter
+++removed several weeks before mucus collection.
The following notes provide a better understanding of the Table 1.
(a) The positivity/negativity of the categories except "no neoplasia" reflects
the well-
known observation that in some individuals these conditions are cancer
precursors
and some are not. The positivity in the category "no neoplasia" is not
explained.
However, some positives still may represent a precancerous condition and,
thus,
not all 13 positives represent a false positive category (see hereinbelow).
(b) No-neoplasia includes also: cancer family history (5, 1+, 4-), irritable
bowel
syndrome (2, 1+, 1-) , hemorrhoids (3, 1+, 2-), and angeodisplasia (1, 1-).
Adenomatous polyp includes adenoma with diverticulosis (1, 1+). Small polyps
include polyps with diverticulosis (2, 2+).
(c) Positivity/negativity of the test in previously removed carcinomas may
reflect the
completeness of the cancer removal.
(d) Inflammatory conditions are considered a risk factor for colorectal
cancer. The
positivity in the test may reflect how far an inflammation has progressed to
an
early stage of cancer.
(e) Percentage and CIs of groups with less than 10 subjects are not
calculated.
(f) This test reclassified 8 cases from "positive" to negative by
distinguishing more
clearly the colour representing "positivity". The 8 cases were in the
following
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categories: 3 "no neoplasia", 1"cancer family history", 1"carcinoma previously
removed", and 3 "small polyps".
Example 3
Mucus reaction in colectomy specimens with colorectal cancer
In order to obtain a sufficient amount of colorectal mucus for chemical
identification of the aldehyde marker, the mucus was collected from segments
of colon
removed from patients with colorectal cancer. At the same time, the presence
of the
marker using Schiff's reagent was established. The results shown herein
confirm the
high sensitivity of the test.
Table 2 shows the results on colectomy specimens obtained from the operation
theatres of several hospitals in Toronto, Ontario, Canada. The specimens were
obtained
as follows.
Colectomy specimens 15 - 20 minutes after surgery were washed with water to
remove blood. The mucus was collected by gently scraping the surface with a
small
spatula without damaging the underlaying mucosa. The scraped mucus was placed
into a
small plastic vial and frozen. For the assay with Schiff's reagent, the vials
were removed
from the freezer, allowed to stand at room temperature for 60 minutes to thaw,
and a
small amount of the mucus on the tip of a spatula was smeared upon the support
and
assayed.
Table 2
Pos. Neg:
Condition Total # % CI # %
Cancer 15 14 93.4 68-100 1* 6.6
*Note that the original color of this mucus specimen was deep green and,
therefore, it
was difficult to determine the color after the reaction with Schiff's reagent.
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Example 4
Isolation and characterization of markers
Mucus obtained from human colectomy specimens as described in Example 3 was
pooled (66 g) and lyophilized for 24 hours to give a semisolid residue (6.0
g). This
residue was consecutively extracted with several solvents and the extracts
with
chloroform-methanol (2:1) and ethylacetate gave positive reaction with
Schiff's reagent.
These extracts were combined and subjected to chromatography on a column of
silica
gel. Chloroform-methanol (7:2.5) afforded a fraction, which after evaporation
to dryness
gave a residue (36.6 mg) positively reacting with Schiff's reagent. After
several
chromatographic separations, a highly positively reacting material (4.2 mg)
was obtained
and further analyzed by NMR spectroscopy. The NMR studies showed that the
fraction
consisted of a mixture of phospholipids, containing both choline (cf the
signal at 83.240
ppm for -N(CH3)3] and ethanolamine. Hydroxyls in the position 1 and 2 of
glycerol were
both esterified with fatty acids in about 40% of the compounds. The remaining
60%
were esterified only at position 2 of glycerol, while at position I was bound
an a,(3-
unsatured ether, identified through a doublet at 85.90 ppm assigned to the
vinylic proton
O-CH=CH-. The a,(3-unsaturated ether is a derivative of a higher molecular
weight
aldehyde, mainly stearaldehyde and palmitaldehyde. The estimate of the ratio
between
the vinylether containing phospholipids (=plasmalogens) and completely
esterified
phospholipids as 3:2 was made on the basis of a comparison of signal
intensities for CH-2
of the glycerol moiety in plasmalogens at 55.58 ppm, the integration of which
correlated
well with the vinylic signal at 55.90 ppm, and in diesterphospholipids at
55.218 ppm.
The aldehydes were identified by comparison with authentic specimens of O-
(2,3,4,5,6-
pentafluorobenzyl oximes of the aldehydes using mass spectrometry and gas-
liquid
chromatography. Both aldehydes exhibited M-20 ions instead of molecular ions,
m/z
415.1 for palmitaldehyde and m/z 443.2 for stearaldehyde.
The O-(2,3,4,5,6-pentafluorobenzyl oximes of the aldehydes were prepared from
O-(2,3,4,5,6-pentafluorobenzyl) hydroxylamine (250 L of 0.05M solution in
sodium
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acetate buffer, pH 5) added to the phospholipid mixture (1 mg in 100 L of
water) which
was vortexed for 1 minute and allowed to react for 30 minutes. Then 1N HCl (10
L) was
added, and the reaction mixture extracted three time with hexane (1 mL). The
combined
hexane extracts were dried over sodium sulfate, evaporated to dryness under a
stream of
5 nitrogen, and the residue redissolved in hexane (50 L). This solution (1 L
injections)
was used in the gas-liquid chromatography-mass spectrometric identification of
the
aldehydes.
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
10 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.
