Classifications

• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
  • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
  • G01N33/57484—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumor, cancer, neoplasia, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides, metabolites
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
  • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
  • G01N33/57407—Specifically defined cancers
  • G01N33/57419—Specifically defined cancers of colon
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
  • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
  • G01N33/57407—Specifically defined cancers
  • G01N33/57446—Specifically defined cancers of stomach or intestine

Definitions

• 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.
• 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.
• 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 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.
• colorectal cancer satisfies each of the following three criteria of a disease considered suitable for a screening program.
• 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 interventions.
• 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.
• 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.
• 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.
• 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.
• HemoQuant porphyrin analysis
• 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.
• 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.
• T-antigen is not expressed by cells in healthy colons, whereas it is expressed by cancer (8).
• 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 Amaranthus caudatus, has been reported to have better specificity for T-antigen than
• 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.
• the invention provides a method for detecting the presence of neoplasia, a precancerous condition or cancer of the large intestine, which comprises:
• aldehyde marker selected from the group consisting of CH 3 (CH 2 ) 14 CHO, CH 3 (CH 2 )j 6 CHO, and precursors thereof; and (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 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.
• 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.
• 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 Schiffs reagent, the latter reagent reacts with aldehydes with high sensitivity to form a purple colored addition compound defined by absorbance at about ⁇ max 560 - 590 nm.
• the utilization of p-rosanilin in the form of Schiffs reagent for detection of aldehydes in colorectal mucus is described in more detail, hereinbelow.
• the method comprises treating said sample with Schiffs reagent and detecting neoplasia or cancer of the colon or rectum based upon the coloration produced at about 560 - 590 nm ⁇ m a ⁇ in said sample by said treatment.
• the specific coloration produced according to the practise of the present invention can be visually seen or detected by spectrophotometric determination at about 560 nm.
• the method does not require the additional step of enzyme treatment for detecting the disaccharide marker beta-D-Gal(l-3)-D-GalNac( ⁇ l-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 obtained by the action of Schiffs 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.
• a purple coloration having a light absorption at about 560 - 590 nm is produced by the Schiffs reagent with the aforesaid aldehydes.
• 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.
• P-rosanilin in water has an absorption at about 538 nm, but is insoluble in dichloromethane.
• Aldehydes of longer carbon chain length with Schiffs Reagent behave similarly as stearaldehyde adduct.
• 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.
• 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.
• 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 Schiffs 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 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 Schiffs 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 Schiffs reagent.
• 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.
• 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.
• purple coloration appears at the place where the mucus was deposited; otherwise the support remains colourless, although some background coloration may develop.
• 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 Schiffs reagent.
• 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 Schiffs 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 may be those of microscope slides which would enable the utilization of the equipment standard for simultaneous development of microscope slides.
• 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.
• a suitable lubricant, such as MUKO, for the rectal examination is chosen from among those that do not react with Schiffs reagent.
• 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 0.1M potassium phosphate buffer (pH 7.0) for 10 minutes, while the tank is gently, mechanically vibrated.
• 0.1M potassium phosphate buffer pH 7.0
• 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 Schiff' 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 Schiffs 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.
• 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.
• the test is negative by reason of no color on the support, it is useful to establish if mucus were deposited in the plate.
• 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. 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.
• 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.
• 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.
• IN 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 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.
• This table shows the results from the endoscopy unit at Wellesley Hospital, Toronto, Ontario, Canada, who agreed to submit themselves to the mucus testing.
• 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+).
• Positivity/negativity of the test in previously removed carcinomas may reflect the completeness of the cancer removal.
• 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.
• the mucus was collected from segments of colon removed from patients with colorectal cancer. At the same time, the presence of the marker using Schiffs 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 Schiffs 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.
• 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 Schiffs 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 Schiffs reagent. After several chromatographic separations, a highly positively reacting material (4.2 mg) was obtained and further analyzed by NMR spectroscopy.
• the ⁇ , ⁇ -unsaturated ether is a derivative of a higher molecular weight aldehyde, mainly stearaldehyde and palmitaldehyde.
• 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 acetate buffer, pH 5) added to the phospholipid mixture (1 mg in lOO ⁇ L of water) which was vortexed for 1 minute and allowed to react for 30 minutes. Then IN HC1 (lO ⁇ 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 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.

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