Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving antigen-antibody binding – specific binding protein...
Reexamination Certificate
1998-04-23
2001-02-13
Le, Long V. (Department: 1641)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving antigen-antibody binding, specific binding protein...
C435S001300, C435S007230, C034S284000, C436S174000, C436S177000
Reexamination Certificate
active
06187546
ABSTRACT:
The present invention relates to a method of isolating cells, especially exfoliated epithelial cells.
Exfoliated epithelial cells consist of cellular components, such as DNA, RNA and proteins, which potentially provide a multiplicity of approaches to identifying and monitoring unhealthy conditions of the epithelium as exemplified by colorectal cancer, polyps and ulcerative colitis. The facile recovery of such cells makes possible the application of a wide variety of test procedures for qualitative and/or quantitative analysis of, for example, DNA alterations, gene expression and enzyme activity.
The early detection of cellular, genetic and other biomarkers of tumorigenesis or malignancy is a critical factor in the prophylaxis and treatment of cancer and other disease conditions. Various cancers including colorectal, lung, oral, pancreatic, bile duct, urinary bladder and some other epithelial tissue cancers occur at substantial incidence (Cancer Incidence in Five Continents, Vol VI, ed. Parkin D. M. et al., IARC Scientific Publications No. 20, IARC, Lyon, France) and have substantial mortality because in many cases tumours are diagnosed too late to be curable due to the absence of non-invasive, inexpensive screening methods for general use. At present many cancer cases are only detected when a tumorous mass or bleeding or other malfunction leads to the use of diagnostic procedures that themselves can only confirm an advanced stage of neoplastic process requiring drastic treatment often with low likelihood of success. Illustrative of this problem is pancreatic cancer in which the early symptoms can be easily confused with those of more common conditions but when the cancer diagnosis is eventually made the average life expectancy is only 3 months. Also illustrative is colorectal cancer which afflicts 5% of the population and kills 3%, but which studies indicate is curable with early intervention. The lack of procedures capable of very early detection of neoplasia at stages before invasion or even before malignancy is evident, although recent methods of detection of cancer-related genetic alterations (Mao L. et al., Cancer Research (Suppl.), 54, 1939s-1940s (1990)) and initial knowledge of a sequence of genetic lesions leading to neoplasia (Fearon E. R. et al., Cell, 67 757-767) provide hope of progress.
Of key importance in the provision of a mass-screening program for early detection and treatment of pre-cancerous and cancerous conditions is the need for simple procedures for obtaining tissue, cell or DNA samples on which diagnostic assays may be performed. For individuals in whom cancer or polyps or a pre-neoplastic lesion has been detected, or are apparently at elevated risk because of genetic predisposition, it is also important to sample the tissue subsequently on a number of occasions to seek evidence of response to treatment or recurrence. Such treatments include those designed to eliminate a tumor, or to induce preventive metabolic changes.
Conventional sampling procedures usually entail taking a tissue biopsy from the patient. However, biopsy procedures are invasive, often require local or general anaesthesia, and at the very least cause considerable discomfort to the patient. Furthermore, except for sampling a tumor or polyp, a biopsy removes a small sample of tissue which is a priori non-representative of the organ or tissue as a whole. Such techniques are therefore unsuitable for routine mass screening.
A screening approach to early detection of colorectal tumors is based on faecal occult blood testing (FOBT). The main advantages of FOBT are its non-invasiveness, simplicity and low cost. However, the presence or absence of blood in stool is not necessarily related to the presence or absence of a tumor. Consequently the method is not free of false-positive results and often fails to detect cancer and especially polyps.
Several studies have indicated that there may be correlation between increased colonocyte proliferation and colorectal cancer risk and/or presence of colorectal tumours (Terpstra O. T. et al., Gastroenterology, 92, 704-708 (1987); Scalmati A. et al., Cancer Res., 50, 7937-7941 (1990); Risio M. et al., Cancer Res., 51, (1991); Al-Sheneber I. F. et al., Cancer, 71, 1954-1959 (1993)). These results, however, often failed to reach statistical significance and were controversial for some patients. The evidence and methods published to date have failed to substantiate increased colonocyte proliferation as providing a basis for a reliable or practical diagnostic test for colorectal cancer risk and/or presence of colorectal tumours.
A method for isolating human cells from homogenised faeces has been reported (Albaugh G. P. et al.,
Int. J. Cancer,
52, 347-350 (1992); Iyengar V. et al.,
FASEB J.,
5, 2856-2859). The reported method employs a homogenised sample of faeces and has been found to be difficult to repeat, and lacks the speed, yields and selectivity to be of use in routine mass screening. Dutta S. K. et al. (AGA Abstracts,
Gastroenterology,
108, (1995) A463 employed this method based on density gradient separation in an attempt to compare the number of colonocytes recovered per gram of homogenised faecal sample in colorectal cancer patients and control subjects. Although there was a trend toward greater recovery in cancer patients (about twice that of controls), the difference failed to reach statistical significance.
A method for isolating metastatic malignant epithelial cells from blood has also been reported (Hardingham et al. Cancer Research, 53, 3455-3458 (1993)). The method employed immunomagnetic beads labelled with a monoclonal antibody specific for epithelial cells to isolate cells, which were analysed for the presence of a point mutation in the K-ras tumour marker gene using PCR. The direct use of immunomagnetic beads would not, however, be expected to be of general applicability to the isolation of epithelial cells from bodily waste products or gastrointestinal fluids in view of the likelihood of non-specific binding interactions between the immunomagnetic beads and material present in the bodily waste product or gastrointestinal fluid.
Extraction of DNA directly from human stool for detection of cancer-associated gene alterations by diluting small portions of homogenized frozen faeces with a cell lysis solution followed by centrifugation of the resultant suspension and purification with powdered glass has also been reported (Sidransky D. et al.,
Science,
256, 102-105 (1992)). Loktionov and O'Neill (
Int. J. Oncology,
6, 437-445 (1995)) reported isolation of DNA from rat stool by incubating faeces with cell lysis buffer and a subsequent phenol-chloroform extraction process. In both these cases only limited amounts of pure DNA for PCR amplification were obtained.
There remains therefore a need for methods of cell sampling which (a) are non-invasive or minimally invasive, (b) are quick so as to recover intact cells, (c) provide a large enough quantity of cells suitable for multiple assays, (d) are selective so as to leave behind irrelevant material that may dilute sample or interfere in assays, (e) are capable of providing a representative collection of cells. A further requirement that underlies many assay methods is that actual cells are obtained rather than a simple recovery of DNA or another cellular component present in the cells of interest.
According to the present invention there is provided a method of isolating cells from a faecal stool the method comprising the step of cooling the stool to a temperature below its gel freezing point and removing cells from the stool whilst maintaining the stool at a temperature below its gel freezing point, such that the stool remains substantially intact.
The present invention is applicable to the isolation of mammalian cells from the stool of any animal (for example humans) with particular application to the removal of cells from the surface of the stool. Humans generally produce soft stools compared to the pelleted stools of some animals, such as rodents. It has been found that cooling
Loktionov Alexandre
O'Neill Ian Kenneth
Le Long V.
Mathews, Collins Shepherd & Gould P.A.
Nguyen Bao-Thuy L.
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