MXPA00012243A - Screening test for early detection of colorectal cancer - Google Patents

Abstract

A method for detecting neoplasia or cancer of the colon or rectum comprising obtaining a sample of colorectal mucus from the rectum of a patient, and detecting the presence of a marker selected from the group consisting of long chain aliphatic aldehydes containing 12 - 20 carbon atoms, particularly CH3(CH2)14CHO and CH3(CH2)16CHO;and plasmalogen-bound precursors thereof. Preferably the method comprises treating the sample with Schiff's Reagent and detecting neoplasia or cancer of the colon or rectum based upon the coloration produced at about 550 - 590 nm in the sample by the treatment. The method does not require the step of adding an enzyme for detecting the disaccharide marker&bgr;-D-Gal(1-3)-D-GalNAc(&agr;1-Thr/Ser) and a saccharide marker containing D-galactose and/or 2-acetamido-2-deoxy-D-galactose.

Description

SIZING TEST FOR EARLY DETECTION OF COLORECTAL CANCER DESCRIPTION OF THE INVENTION This invention relates to a simple screening test for colorectal cancer wherein a marker is detected in the rectal mucus. More particularly, this marker is detected in the mucus deposited on a support using a Schiff reagent. Colorectal carcinoma is the second most common cause of cancer mortality in men and women, causing almost one third of all malignancy-related deaths in North America. It has been estimated that as recently as 6% of Canadians and Americans developed malignancies in the small intestine, and more than 50% of them will die within 5 years of diagnosis. Many authorities believe that colorectal cancer can only be controlled by preventive measures (1) since there are no realistic prospects to significantly improve the rate of cure once the cancer has spread beyond the intestinal wall. Primary prevention, that is, warning the development of the tumor by altering biological risk factors, is not feasible since very little is understood of the etiology of the disease. Alternatively, secondary prevention measures, i.e. detection in a treatable attic state, may be possible if an effective screening test is available. Indeed, the neoplasms of the large intestine have 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) while once the true stage of cancer is reached, and showing symptoms, the mortality ratio is more than 50% . The removal of neoplasms from the intestine in its early, asymptomatic state can be done by non-surgical endoscopic polypectomy, without any significant risk. On the other hand, this requires at least four to six years before an adenomatous polyp reaches the stage of cancer, so there is an incredible opportunity to detect these neoplasms in their treatable stage. Recent clinical studies document a decrease in mortality as a result of screening for colorectal cancer, as predicted by these theoretical considerations. The problem to date has been that polyps can be easily detected only by endoscopy. In this way, colorectal cancer satisfies each of the following three criteria of a disease considered adequate for a screening program. First, it is a relatively common condition with serious consequences. Second, curative treatment is available when it is detected at an early stage, that is, polypectomy of cords through surgical segmental bowel resection. Third, the prevalence is high enough to justify the costs 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 stage sufficiently large to allow curative treatment. It is not designed to necessarily diagnose a disease, but to determine that asymptomatic individuals apparently free of disease can undergo diagnostic interventions. The ability of a screening test to distinguish those who are in danger after the evaluation of those who are not expressed in epidemiological terms. The term "sensitivity" is defined as the proportion of sick individuals who have a positive test, that is, the proportion of true / relative positives to all people with the disease. The "specificity" is the proportion of free subjects to disease who have a negative test, that is, the proportion of true / relative negatives to people without the disease. The term "positive predictive value" is the proportion of positive tests due to the disease, that is, the proportion of true positives / relative to all positives. Almost always, sensitivity and specificity must be managed with each other. Intuitively, it seems in one way to design a screening test for a fatal disease to optimize sensitivity, in order to detect as many individuals with the disease as possible. It is emphasized, however, that optimization sensitivity comes with a risk of reducing specificity to such an extent that unacceptably high costs, poor compliance, and "flooding" of diagnostic facilities result. On the other hand, the positive predictive value, which is particularly useful expression of the value of a screening test, depends critically on the specificity and on the prevalence of the disease in the sifted population. It has been pressed that the efficacy of a screening test can be properly evaluated only by random controlled tests. In the case of cancer, it is not enough to show that life is prolonged when the malignancy is detected by a positive screening test, compared when the tumor is diagnosed after the development of symptoms. Rather, it should be shown that screened individuals have a death rate lower than malignancy than similar individuals not enrolled in such screening program. A particularly fallacious presumption is that the predictive value of a screening test is the same in a hospitalized population with advanced disease, in which the test is initially tested, since it is in a healthy population with minimal early disease, to which the Proof is usually proposed. Screening methods in current population Endoscopic methods, such as sigmoidoscopy or full-length colonoscopy, are diagnostic rather than screening techniques, although sigmoidoscopy is sometimes used for screening. The current routine method of screening for colorectal cancer in the general population is to look for occult blood in the fecal matter (3). The current techniques, for example, HemOccult II, involve swabbing a sample of fecal material on paper impregnated with guaiac which, after treatment with hydrogen peroxide containing developer, exhibits blue color if blood is present (hemoglobin). After almost two decades of experience with this methodology, it has become clear that even in centers of experiments, the sensitivity is less than 50% for curable neoplasms, and that the positive predictive value approaches, at best, only 40%. % in a clinical population. An update of the major scale (n = 97, 205) University of Minnesota, Minnesota, United States, prospective test indicates a positive predictive value for colorectal cancer of only 2.2% (4). Additionally, factors such as medications, multiple diet constituents, delays in specimen handling, fecal hydration variability, and storage of test materials, commonly confrontational results. The analysis of one of the three randomized controlled studies assessing the HemOccult value suggests comparable mortality ratios in the screened and control populations (5). New methods to detect occult blood, for example methods based on either porphyrin analysis [HemoQuant] or a specific antibody for human hemoglobin, improve these results. However, three limiting problems remain, probably unresolved. These are that the colorectal malignancies cover blood only intermittently, over the gastrointestinal tract the bleeding can take the results of false positives, and multiple lesions in the large intestine, apart from the colorectal neoplasms, commonly bled. Such injuries include hemorrhoids, verticulated d, ulcers, and vascular ectasies. Compliance in unselected populations has been estimated to be less than 30%, at least partially since the technique requires patients to self-swab their fecal matter on a strip or tape, a task that most people find not unpleasant, but also technically difficult. Despite this, HemOccult continues to be widely used since the American Cancer Society has recommended occult blood tests annually for all individuals over 50 years of age, arguing that even an imperfect test will save many lives. It is implicit in all the arguments about the value of HemOccult that any improvement in screening techniques for bowel malignancies can have a dramatic impact on mortality rates of colorectal cancer, since screening for hidden blood even in the present form leads to reduced the mortality of colorectal cancer (6). Experimental screening methods (i) sieving for colorectal cancer by faecal DNA analysis (7). This is based on the presence in fecal matter of neoplastic cells scattered in large numbers in the colonic lumen. In the beginning, a mutation which is common in neoplasms can be detected with high precision by analyzing DNA from these cells. Currently, the most common mutation is the mutation of the K-ras oncogene present in 40% carcinomas and colorectal adenomas. The screening for the K-ras gene can, therefore, detect, at its best, only 40% of all neoplasms. This methodology is currently technically complex and expensive. (ii) Screening for the presence of colonic mucin from a disaccharide related to cancer, D-Galp (ßl-3) -D-GalpNAc (al, Ser / Thr), antigen T- (Thomsen-Friedenreich), since it is It is widely known that the T antigen is not expressed in healthy colon cells, while it is expressed by cancer (8). (a) monoclonal antibodies and lectins. It has been shown that monoclonal antibodies raised against the 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 less specificity. Amaranth, a lectin from Amaranthus caudate, has been reported to have better specificity for the T antigen than PNA. Neither amaranthina nor PNA bind to histological sections of normal mucosa, but both bind to mucin in Goblet cells of tumors and certain polyps, and in the transitional mucosa. The visualization of the link uses fluorescently labeled antibodies and lectins (9). (b) Galactose oxidase test. Antigen T is also reported to be colorimetrically detectable after oxidation of OH-6 of galactose using galactose oxidase and visualization of the resulting aldehyde with Schiff's reagent, US Pat. No. 4,857,457, issued August 15, 1989; U.S. Patent 5,348,860, issued September 20, 1994; and Patent of the States United 5,162,202, issued November 10, 1992, to Shamsuddin et al. In contrast to tests that use lectins, this test is performed on mucus samples obtained by digital rectal examination and smear on a support.

This system demonstrates a sensitivity of 74% and specificity of 50% for colorectal neoplasms, ie adenomatous polyps and cancer, in a study with only a false negative result among 59 patients with cancer.

Subsequently, a number of reports of basically the same test have appeared with sensitivity in the range of 35% to 100% and specificity in the range of 15% to 76%. Some researchers found that the test is more sensitive, but less specific, than HemOccult. The lower specificity has been confirmed with the positivity of the test in individuals with certain inflammatory conditions, such as diverticulitis and ulcerative colitis (10). In contrast to the prior art mentioned above, a colorectal mucus test does not require detection of the disaccharide marker beta-D-Gal (l-> 3) -D-GalNAc and a saccharide label containing D-galactose and / or 2 -acetamido-2-deoxy-D-galactose has been described in the US Pat. 5,416,025 assigned to Krepinsky et al., Issued May 16, 1995. In this method, a sample of colorectal mucus is treated with Scriff reagent, without a step of adding an enzyme to detect the aforementioned disaccharide marker, and detecting the Change of color in the sample. The method described in U.S. Patent No. 5,416,025 demonstrates a sensitivity of 92% for colorectal cancer from a test with 25 patients with cancer. However, the specificity is somewhat compromised since several shades of pink coloration are frequently obtained and result in false positives. Although the screening test described in U.S. Patent No. 5,416,025 provides a significant improvement over prior art methods mentioned above, 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 test which also reduces the probability of false positive or false negative readings. List of references The present specification refers to the following publications, each of which is expressly incorporated herein by reference. Publications 1. Lieberman D. A .: Targeted colon cancer screening: A concept whose time has almost come. Amer. J. Gastroenterol. 1992, 8", 1085. 2. Eddy DM: Screening for colorectal cancer Ann. Int. Med. 1990, 113, 3U 3. Rex DK Lehman G., A., Ulbright TM, Smith JJ, Pund D. C , 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 Amer. J. Gastroenterol 1993, 88, 825 4. Mandel JS, Bond JH, Bradley M., Snover DC, Church TR, Williams S., Watt G., Schuman LM, Ederer F., Gitsen V .: Sensitivity, specificity, and positive predicti-vity of the Hemoccult test in screening for colorectal cancer, Gastroenterol, 1989, 97, 597. 5. Selby JV, Friedman GD, Quesenberry Jr. CP, Weiss NS: Effect of fecal occult blood testing on mortality from colorectal cancer Ann. Intern. , 118, 1. 6. Mandel JS, Bond JH, Church TR, Snover DC, Bradley GM Schuman LM, Ederer F.: Reducing mortality from colorectal cancer b and 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 occuring with cellular differentiation and malignant transformation. Proc. Nati Acad. Sci. USA 1982, 79, 2051. 9. 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 crypctic T) antigen . J. Biol. Chem. 1989, 264, 16123. l.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. ll.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. Dog. 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 Patent of the United States No. 4,857,457, Shamsuddin et al. August 15, 1989. United States Patent No. 4,762,800, Rettig et al., August 9, 1988. United States Patent No. 4,863,854, Mattes et al. September 5, 1989. U.S. Patent No. 4,962,187, Pant, Oct. 9, 1990. U.S. Patent No. 5,073,493, Yamashina December 17, 1991. U.S. Patent No. 5,008,184, Linnane, April 16, 1991.

U.S. Patent No. 5,162,202, Shamsuddin, November 10, 1992. U.S. Patent No. 5,348,860, Shamsuddin, September 20, 1994. U.S. Patent No. 5,416,025, Krepinsky et al., May 16, 1995. It is an object of the present invention to provide a tool for screening asymptomatic persons for cancer of the large intestine and rectum. It is a further object of the present invention to provide an improved screening test for detecting neoplasms of the large intestine and rectum prior to the development of a bleeding cancer. It is still a 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 observed 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 colon cancer or rectal condition thereof, and detecting the presence of a marker selected from the group of large chain aliphatic aldehydes containing 12 -20 carbon atoms, optionally, containing olefinic groups; more particularly aliphatic aldehydes containing C16-C18; and plasmalogen binding 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 mucus sample from the large intestine of a patient's rectum; (b) testing the sample to detect an aldehyde tag selected from the group consisting of CH3 (CH2) 14CHO, CH3 (CH2)? 6CHO, and precursors thereof; and (c) detecting neoplasia, precancer or cancer of the large intestine based on 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 linked to plasmogen. The marker is preferably selected from the group consisting of octadecanal, commonly referred to as stearaldehyde, of the formula CH 3 (CH 2) 6 6CHO, hexadecanal, common name palmitaldehyde, of the formula CH 3 (CH 2) ßCHO, and 9-octadecenal, common name olealdehyde of the formula CH3 (CH2) 7CH = CH (CH2) 7CHO. In preferred embodiments of the invention, knowledge of the structures of the aldehyde markers allows observations of the presence of the aforementioned aldehydes in colorectal mucus using properties specific to the aldehyde group, for example, by polarography or by using reagents that react specifically with compounds that they form the aldehyde group detectable by their resulting suitable properties, such as color, for example, specific spectral properties, fluorescence, mass spectral, chemiluminescence 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, upon their release, react immediately with the reagent. The excess of the unreacted reagent is more preferably removed, for example by repeated washing with water and / or buffers. Many known aldehyde detection 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, finished addition compounds with easily detectable properties, such as fluorescence or color. Examples, of such compounds containing the amino group are in the group of dyes based on aniline. P-Rosaniline is a particularly suitable dye, since after being transformed by reaction with a sulfide or analogs in aqueous hydrochloric acid in colorless Schiff reagent, the latter reagent reacts with aldehydes with high sensitivity to form a purple-colored addition compound defined by absorbance at approximately 560-590 nm. The use of p-rosaniline in the form of the Schiff reagent for detection of aldehydes in colorectal mucus is described in more detail, hereinafter. Preferably, the method comprises treating the sample with the Schiff's reagent and detecting neoplasia or cancer of the colon or rectum based on the staining produced at approximately 560-590 nm max in the sample by the treatment. The specific coloration produced according to the practice of the present invention can be visually observed or detected by spectrophotometric determination at approximately 560 nm. More preferably, the method does not require the additional step of enzyme treatment to detect the disaccharide marker beta-D-Gal (1-3) -D-GalNac (al-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 the Schiff's reagent on mucus components collected from individuals with neoplastic disease of the right color can be visually observed or measured spectrophotometrically to better indicate true positives and reject false positives. We have discovered that the mucus collected from such individuals contains detectable quantities of the large chain fatty aliphatic aldehydes CH3 (CH2) i4CHO and CH3 (CH2) 66CHO, and the olefinic aldehyde CH3 (CH2) 7CH = CH (CH2) 7CHO, per se, c are linked within plasmalogens present in the mucus and released from it by the acid of the Schiff reagent. It has been found herein that a purple coloration that has a light absorption at about 560-590 rs ?. it is produced by the Schiff reagent with the aldehydes mentioned above. It has been found that mucus from neoplastic disease-free individuals does not provide the visually identifiable color spectrum observed in the mucus of individuals with neoplastic disease. Additionally, colorectal mucus has been found to contain basic compounds that cause the Schiff reactive to reverse the original dye, p-rosaniline.; and produce a reddish pink coloration that has a light absorption at 538 nm? max. It is often difficult to distinguish this undesirable coloration from the search for weak purple coloration, without the need for a color chart, and this, consequently, can lead to an increase in false positives and, thus, reduced specificity of the assay. . It has further been found that although a number of true 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 with the assay of the present invention. invention, if, preferably, sufficient water wash of the mucus is carried. In this way, in contrast to the prior art indicated above, if water washing and strict observation of the purple color at about 560-590 nm as the appropriate indicator of the presence of the molecular weight aldehydes and, thus, the Presence of a precancerous condition or cancer is maintained, a substantial reduction of false positives is obtained, and consequently a substantial increase in the specificity of the test. The purple coloration produced according to the practice of the invention due to the presence of the aforementioned large chain aliphatic fatty aldehydes is distinguishable from the various shades of pink and red coloration caused by other substances present in the colorectal mucus which remains in the mucus after water wash. A color chart attached to each equipment helps in the proper identification of the purple color, even by non-entrants, and thus allows an operator to maintain the high specificity of the test. It should be noted that the value-specific purple coloration in the practice of the invention does not develop with p-rosaniline alone, although the Schiff's reagent per se is prepared from p-rosaniline. It has been found that the exact position of the Schiff reagent of the large chain aldehyde adduct is dependent on the solvent. It has been found that the Schiff reagent adduct stearaldehyde in dichloromethane exhibits a relatively broad maximum absorption at about 590 ^ -nm 'with one arm at about 555 + 1 nm. This ac c: o in ethanol shows an absorption in approximately 547 nr. with one arm at approximately 578 nm. In the latter solution, the color changes from purple to red which is believed to be due to the instability of the adduct in ethanol. It is insoluble in water. P-rosaniline in water has an absorption at about 538 nm, but is insoluble in dichloromethane. The large carbon chain length aldehydes with the Schiff reagent behave similarly as an adduct of stearaldehyde. For example, the adduct of miristaldehyde in dichloromethane shows a maximum absorption at about 586 nm with one arm at about 556 nm. The formaldehyde adduct in water gives a broad flat maximum extension of about 560 - about 593 nm. The important advantage of rectal mucus test, compared with 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 aligned along its entire length with mucus, a viscoelastic gel composed of water, electrolytes, organic chemicals, such as nucleosides and nucleotides, amino acids, peptides, lipids, lipids that include phospholipids and oxidation products of lipids, and large molecular weight glycoproteins (mucins), as well as cells and sloughed bacteria, which are movable throughout the intestine, it is believed that the rectal mucus contains mucus from the entire colon, i.e., the r.oco secreted by a neoplastic tissue flows along the intestine in the rectum in which the spot is shown. A general method of use in the implementation of the invention is as follows. A sample of mucus obtained by a trained physician or nurse using a gloved finger lubricated with MUKO or a similar lubricant which does not trigger any color change in the Schiff's reagent during the digital rectal examination of a sifted individual is deposited in a suitable water insoluble substrate or support, such as a pad or a disc. Suitable support materials are prepared from, for example, glass microfibers, some polymer fibers such as polyester and cellulose fibers or modified cellulose fibers. The support may or may not be pretreated with antioxidants such as BHT (butylated hydroxytoluene) or BGA (butylated hydroxyanisole). The following procedure is preferably employed. The mucus sample is deposited on a support as described hereinafter, retained therein for approximately 90 minutes before raising it, or, if taken from a freezer, thawing is allowed for approximately 90 minutes. Subsequently, the support that carries the mucus is increased in potassium phosphate buffer 0.1 M, generally for approximately 10 minutes, washed twice with water for 2 minutes, air-dried for 15 minutes to remove excess water, such about 2 minutes, it is washed briefly with distilled water, and dried in the air. A positive reaction is classified when a purple color appears on the filter within 20-25 minutes after removal of the Schiff reagent. If a specimen does not produce any coloration, it is either due to the absence of the long-chain aliphatic aldehydes or plasmalogen precursors in the mucus, or because the mucus has not been picked up by the gloved finger and, therefore, not deposited in the support. To distinguish between these two possibilities, a negative test medium with 0.5% periodic acid solution is treated for 5 minutes, rinsed with water, stained with Schiff's reagent for 5 minutes and rinsed again. When the mucus lacks the present marker, the purple coloration remains colorless, although some background coloration may develop. During the practice of the process according to the invention, other colors than purple can be observed, in particular those that lead to pink and red tones. Such colors reflect the presence of basic substances that release the original red-pink p-rosaniline dye from the colorless Schiff's reagent. Since the basic substances noted herein are usually water-soluble, these variations usually indicate that the washing of samples prior to treatment with the Schiff's reagent is incomplete. It is known that the properties of the Schiff reactant vary according to the combination of several isomers present in commercial p-rosaniline preparations and according to the method of preparation of the same Schiff reagent. However, in contrast to the technique mentioned hereinabove, U.S. Patent No. 5,416,025 assigned to Krepinsky et al., These variations do not exert a significant influence on the test due to knowledge of the aldehyde nature of the marker and the chemical properties of the adduct between the aldehydes and the Schiff's reagent prepared using a suitable procedure. The preferred method i for the preparation of an appropriate Schiff reagent is described hereinafter. To obtain reproducible results with maximum sensitivity and stability, it is desirable to allow the reagent to mature for 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 equipment comprising, for example, a container such as a package, carton, tube, box, roller, ribbon or other capsule-like object comprising a water-insoluble substrate capable of of absorbing colorectal and wettable mucus in water and aqueous solutions and Schiff's reagent. The substrate can generally be exposed through a suitable circular opening of, say, for example, 1.0-1.3 cm in diameter between two hard, rectangular plastic plates, rigidly sealed using a double-sided tape. The dimensions of the sealed assembled plates can be those of slides that can allow the use of the standard equipment for simultaneous development of slides. In operationA doctor or a nurse, for example, smears a specimen of mucus on the surface of the support on the plate. The plates are transferred to a laboratory, where they are processed in batches the size of which is determined by the equipment used in the practice of the test, for example, of plates of ten, as described later in the present. The plates are discharged after the results are read. A method such as a screening test for early detection of neoplasia of the large intestine and rectum is described hereinafter. In the holder secured in the plates, as described hereinabove and convenient for driving in a doctor's office, a mucosa specimen obtained during rectal examination is made. A suitable lubricant, such as MUKO, for rectal examination is chosen from those that do not react with the Schiff's reagent. For processing, the following method has been found suitable. The individual plates that carry specimen of mucus in smears are placed in a holder that carries ten plates. The fastener is immersed in a container containing 0.1 M potassium phosphate buffer (pH 7.0) for 10 minutes, while the tank is vibrated gently and mechanically. When the vibration is stopped, the tank clamp is raised, and the clamp is subsequently submerged in a tank containing distilled water and vibrated gently for another two minutes. The washing with water is repeated once, the bra is then raised above the tank, and excess water in the tank is allowed to drain for ten minutes. The fastener with the plates is submerged subsequently in another tank containing the Schiff reagent described hereinafter, vibrated gently for 2 minutes, then taken again and washed three times with distilled water when immersed in a tank containing water by 2 minutes in each case. The fastener with the plates is then air dried and sorted when the purple color appears on the support within 20-25 minutes. The minutes are counted from the elimination time of the Schiff reagent. The color is compared to the color chart, and colors other than purple are counted as negative. Fecal matter deposited on the support along with the mucus may cause an undesired 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 transformation from occurring, pretreatment of the mucus-free support can be performed prior to the mucus deposition with 0.1% solution of an antioxidant, such as, for example, BHT in 95% ethanol or BHA. If the test is negative because of no color in the support, it is useful to establish if mucus was deposited on the plate. To achieve this goal, the specimen is then treated with periodic acid Schiff's reagent to determine if the mucus was deposited on the plate. If the mucus is present, a purple color appears. The smear often shows a slightly yellow color when mucus is present; the colorless deposit usually indicates that only a colorless lubricant is deposited. It should be noted that a weakly positive test can be expected if only a small amount of mucus is present in the support, and, in this way, 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 that increases the risk of neoplasia. For example, a segment of inflamed bowel may be transformed into a preneoplastic condition, and this may be detected by the test. The high sensitivity of the test for neoplasms can reduce the number of patients suffering colonoscopies since they have rectal bleeding, anemia deficient iron unexplained or a first degree in relation to the tumor. Example 1 Preparation of Schiff's Reagent Distilled water (220 ml) is boiled, removed from the heat source and p-rosaniline (0.4 g) is added. The mixture is stirred well and boiled again for 5 minutes, cooled to 50 ° C, and the solution is filtered through a folded paper filter. 1 N 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 light strawberry-colored solution is obtained, to which charcoal is added (NORIT, 300 mg) and the mixture is stirred vigorously for 1.5 minutes. Subsequently, the solution is filtered through a double paper filter in 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 consistently been very high since little cancer is undetected. However, the specificity measured in the clinical control population is imprecise in patients, who at the time of the test, have not clinically detected neoplasms but have some other non-specific conditions, which may well predispose to the development of cancer in the future. . Previously, it is shown that the proportion of false positives among volunteers, healthy young people - not patients - does not exceed 10.6%. Inflammatory conditions of the large intestine are considered to increase the risk of cancer. A segment of inflamed intestine can, 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 the mucus of a group of patients from the endoscopy unit of Wellesley Hospital, some of these patients are diagnosed with putative precancerous condition and colorectal cancer. Table 1 This table shows the results of the endocopy unit at Wellesley Hospital, Toronto, Ontario, Canada, who agree to submit them to the mucus test. Positive Negative Condition To #% 95% #% 95% ta Cl * Cl * i Diverticular disease 4 33 1 Adenomatous polyp 3 3 Polyp < 1 cm + 21 10 48 26-70 11 5,2 29-74 Large polyp > 1 cm ++ 1 1 No neoplasia 45 13 29 32 71 56-84 Cancer 5 5 After elimination of 5 3 2 cancer *** Crohn's disease 2 1 1 Ulcerative colitis 4 3 1 * CI: confidence interval of percentage of positive or percentage of negative. + less than 1 cm in diameter ++ more than 1 cm in diameter +++ several weeks removed before the collection of mucus. The following notes provide a better understanding of Table 1. (a) The positivity / negativity of the categories except "no neopiasia" reflects the well-known observation that in some individuals these conditions are cancerous precursors and are not. The positivity in the categolria "no neoplasia" is not explained. However, some positives can still represent a precancerous condition and, thus, not all 13 positives represent a false positive category '(see later in this). (b) No neoplasia also includes: historija in the cancer family (5, 1+, 4-), irritable bowel syndrome (2, 1+, 1-), hemorrhoids (3, 1+, 2-), and angeodisplasia (1, 1-). The adenomatous polyp includes adenoma with diverticulosis (1, 1+). Small polyps include polyps with diverticulosis (2, 2+). (c) The positivity / negativity of the test in previously eliminated carcinomas may reflect the totality of the cancer elimination. (d) Inflammatory conditions are considered a risk factor for colorectal cancer. Positiveness in the test may reflect how much inflammation has progressed to an early stage of cancer. (e) The percentage and Cl of groups with less than 10 subjects is not calculated. (f) This test is reclassified from 8 cases of "positive" to negative by distinguishing more clearly the color that presents "positivity". The 8 cases are in the following categories: 3"no neoplasia", 1"history in the cancer family", 1"carcinoma previously eliminated" 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 is collected from colon segments removed from patients with colorectal cancer. At the same time, the presence of the marker is established using the Schiff reagent. The results shown here confirm the high sensitivity of the test. Table 2 shows the results in colectomy specimens obtained from the operating amphitheater of several hospitals in Toronto, Ontario, Canada. The specimens are obtained as follows. Collectomy specimens 15-20 minutes | After surgery they are washed with water to remove blood. The mucus is collected by gently scraping the surface with a small spatula without damaging the lower mucosa. The scraped mucus is placed in a small plastic vial and frozen. For the test with the Schiff's reagent, the vials are removed from the freezer, allowed to stand at room temperature for 60 minutes to thaw, and a smear of a small amount of mucus is made on the tip of a spatula on the support and rehearse Table 2. Positive Negative Condition To #% 95% Cl * #% ta 1 Cancer 15 14 93.4 68-100 1 * 6.6 * Note that the original color of this mucus specimen is dark green and, therefore, it is difficult to determine the color after the reaction with the Schiff reagent. Example 4 Isolation and label characterization The mucus obtained from human colectomy specimens is grouped as described er. Example 3 (66 g), and lyophilized for 24 hours to give a semi-solid residue (6.0 g). This residue is extracted consecutively with several solvents and the extracts with chloroform-methanol 82: 1) and ethyl acetate give a positive reaction with Schiff's redox. These extracts are combined and subjected to chromatography on a silica gel column. The chloroform-methanol (7: 2.5) produces a fraction, which after evaporation to dryness gives a residue (36.5 mg) reacting positively with the Schiff reagent. After several chromatographic separations, a material that reacts positively in high form (4.2 mg) is obtained and further analyzed by NMR spectroscopy. NMR studies show that the fraction consists of a mixture of phospholipids, which contain both choline (see The signal at 63,240 ppm per-N (CH3) 3] and ethanolamine.The hydroxyl at position 1 and 2 of glycerol are both esterified with fatty acids in approximately 40% of the compounds, the remaining 60% is esterified only in position 2 of glycerol, while in position 1 it is bound in a, ß-unsaturated ether, identified through a doublet 55.90 ppm assigned to the vinyl proton 0-CH = CH- The α, β-unsaturated ether is a derivative of an aldehyde of higher molecular weight, mainly stearaldehyde and palmitaldehyde The estimate of the ratio between the vinyl ether containing phospholipids (= plasmamalgens) and completely esterified phospholipids as 3: 2 is made on the basis of a comparison of signal intensities for CH-2 of the glycerol portion in plasmalogens at 55.58 ppm, the integration of which correlates well with the signal vinyl at 55.90 ppm, and at diesterfosfolipids at 55.218 ppm. Aldehydes are identified by comparison with authentic specimens of 0- (2, 3, 4, 5, 6-pentafluorobenzyl oximes of the aldehydes, using mass spectrometry and liquid gas chromatography.) Both aldehydes exhibit M-20 ions instead of molecular ions. , m / z 415.1 for palmitaldehyde and m / z 443 J2 for stearaldehyde The 0- (2, 3, 4, 5, 6-pentanofluorobenzyl oximes of the aldehydes are prepared from = - (2,3,4,6-pentafluorobenzyl) hydroxylamine (250 μl of solution at 0.05 m in sodium acetate buffer, pH 5) added to the mixture of phospholipid (1 mg in 100 μL of water) which is vortexed for 1 minute and allowed to react for 30 minutes. Then add 1 N HCl (10 μl), and extract the reaction mixture three times with hexane (1 ml), dry the combined hexane extracts in sodium sulfate, evaporate to dryness under a stream of nitrogen, and The residue is redissolved in hexane (50 μl) This solution (1 μm injections) l) Mass spectrometry of the aldehydes is used in the identification by liquid gas chromatography. Although this description has been described and illustrates certain preferred embodiments of the invention, it is understood that the invention is not restricted to those particular embodiments. Rather, the invention includes all modalities which are functional or mechanical equivalents of the specific and characteristic embodiments that have been described and illustrated.

Claims (10)

1. CLAIMS 1. A method for detecting neoplasia, a precancerous condition or cancer of the colon or rectum comprising obtaining a sample of colorectal mucus from a patient's rectum and detecting the presence of a marker selected from the group consisting of large chain aliphatic aldehydes that they contain 12-20 carbon atoms and plasma-linker precursors thereof.
2. 2. A method according to claim 1, characterized in that the aliphatic aldehyde is selected from CH3 (CH2) 14CHO and CH3 (CH2) CH0.
3. 3. A method according to claim 1, characterized in that it comprises: (a) obtaining a sample of mucus from the large intestine of a patient's rectum; (b) assaying the sample to detect an aldehyde tag selected from the group consisting of CH3 (CH2)? 4CHO, CH3 (CH2) idCHO, and plasminogen binding precursors thereof; e (c) indicate neoplasia, precancer or cancer of the large intestine based on the presence of the aldehyde detected in the mucus.
4. 4. A process according to any of claims 1 to 3, characterized in that the marker is detected immunochemically.
5. 5. A process according to any of claims 1 to 4 characterized in that the marker is detected quantitatively.
6. 6. A method according to any of claims 1 to 5, characterized in that it comprises treating the sample with the Schiff's reagent and detecting neoplasia, a precancerous condition or cancer of the colon or rectum, based on the coloration produced at 550-590 nm in the sample for the treatment.
7. 7. A method in accordance with the claim 6, characterized in that the coloration is compared visually with the colors of a group of standard colors of a color card.
8. 8. A method according to claim 6, characterized in that the coloration is measured by spectrophotometric absorption at 550-590 nm.
9. 9. A method according to any of claims 6 to 8 wherein the sample is treated with the Schiff reagent without a step of adding an enzyme to detect the saccharin marker β-D-Gal (1-3) -D -GalNac (al-Thr / Ser) and a saccharide marker containing D-galactose and / or 2-acetamido-2-deoxy-D-galactose. A method according to any of claims 6 to 9, characterized in that it further comprises absorbing the mucus in a water insoluble substrate and washing the absorbed mucus with an aqueous solution to selectively remove the lower chain aliphatic aldehydes containing less than 12 carbon atoms of the substrate.