Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving antigen-antibody binding – specific binding protein...
Reexamination Certificate
1998-11-10
2001-04-24
Bansal, Geetha P. (Department: 1642)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving antigen-antibody binding, specific binding protein...
C435S006120, C435S007100, C435S040500, C435S040510, C435S040520, C435S068100, C436S503000, C436S535000, C436S043000, C530S387100
Reexamination Certificate
active
06221623
ABSTRACT:
FIELD OF THE INVENTION
The field of the invention is biochemical methods for screening for cervical dysplasia and cancer in exfoliated cells.
BACKGROUND OF THE INVENTION
Human cervical epithelium can elaborate a series of progressive neoplastic changes known as cervical dysplasia or cervical intraepithelial neoplasia (CIN), which are precursors to invasive cervical cancer. The designations CIN I, II and III refer to mild, moderate, and severe dysplasia/carcinoma in situ, respectively. This histological grading scheme is based largely on the extent to which the thickness of the epithelium is replaced by mitotically active cells with enlarged, hyperchromatic nuclei [1]. Untreated, a subset of dysplastic lesions will advance to cancer, with a frequency that increases dramatically in CIN III lesions [2]. Infection with the human papillomavirus (HPV) is strongly correlated with the development of cervical cancer [3, 4]. HPV is present in over 90% of CIN lesions [5], 95% of cervical cancer [4], and 93% percent of anal squamous cancers in women [6].
In many developing countries, cervical cancer is the most common cancer (excluding skin) in women and the major cause of cancer-related deaths in women [7]. In the United States, abnormalities on Papanicolaou, or “Pap” smears are detected in millions of women annually, resulting in an estimated annual cost of $6 billion for patient evaluation and treatment [8]. The success of Pap smear screening is reflected by the fact that there are only approximately 15,000 new cases of cervical cancer annually in the United States. Indeed, a significant body of evidence supports the observation that population-based Pap smear screening can significantly reduce the incidence of cervical cancer between 3 and 10-fold [9]. Nevertheless, at least 10% of dysplasia cases are diagnosed as negative by Pap smear (false negative).
The Pap smear test consists of collecting cells from the cervix and vagina, spreading them onto a glass slide, fixing and staining the cells, and analyzing them under a microscope. Cytological features that distinguish dysplastic cells, such as enlarged hyperchromatic nuclei and increased nuclear/cytoplasmic ratio, are identified by visually scanning the entire slide. While the success of the Pap smear at detecting cervical precancers and cancers is irrefutable, the assay has several limitations: It is a sampling technique in which typically fewer than half of the cells collected from the cervix are transferred to the slide for analysis, contributing to false negative test results. It is dependent upon human observers to read each cell on the slide, generating another source of false negative results due to observer error. It has a relatively long turn-around time, ranging from several days to weeks, impeding effective followup in much of the world. The test is relatively labor-intensive and requires trained personnel, making it unavailable as a screening test in many areas. Some of these limitations have recently been addressed by new technologies available in North America and Europe. Cytyc Corp has introduced an improved collection and slide preparation technique called the ThinPrep Test in which cells are collected into a liquid fixative rather than directly applied to a slide in the field, and delivery of the cells to the slide is performed mechanically, generating a random distribution of cells on the slide more representative of the original population of cells collected. The slides generated by this technology are easier to interpret microscopically, making the test more sensitive to abnormalities. Two companies have recently marketed computers based on neural network programming which digitally analyze cells on Pap smears and identify cells suspicious for dysplasia and cancer: these cells are then reinterpreted by a human observer. This technology is intended to reduce the false negative rate, and is currently only recommended for slides that have previously been analyzed in the conventional fashion and read out as negative.
Similarly, other attempts to identify noncytological markers of cervical dysplasia have only confirmed the need to supplement such assays with conventional cytological examination such as Pap smear to effect clinical diagnosis with tolerable accuracy, e.g. Kyo S et al. (1997) Application of telomerase assay for the screening of cervical lesions. Cancer Res 57(10), 1863-1867; Pillai MR (1996) The presence of human papillomavirus-16/-18 E6, p53, and Bc1-2 protein in cervicovaginal smears from patients with invasive cervical cancer. Cancer Epidemiol Biomarkers Prev 5(5), 329-335. Accordingly, an object of the invention is to provide a screening method amenable to wider, preferably ready, international availability, that can detect premalignant cervical disease before it becomes invasive without the need for any supplemental conventional cytological examination.
SUMMARY OF THE INVENTION
The invention provides methods and compositions for primary screening for cervical dysplasia. The methods involve measuring a biochemical marker in each of a population of cells derived from convenient, superficial swabbing, sponging, scraping or lavage of superficial epithelial cells from the cervix, wherein the marker indicates the presence of cervical dysplasia in the sample, and scoring the results of the measuring step for cervical dysplasia (i.e. ascertaining whether or not the marker is present) in the patient in the absence of any cytological examination. The methods obviate the need for any cytological examination, providing a primary diagnostic clinical screen without the need for time, labor and material intensive resources such as microscopy equipment, etc. The marker or diagnostic marker threshold is present/exceeded in at least 65%, preferably at least 75%, more preferably at least 85% of high grade dysplasia cases.
Accordingly, the invention provides means to detect precancerous or cancerous conditions of the cervix by a label (e.g. fluorescent or colorimetic) that is amenable to low-technology analysis and/or automated, machine-readable analysis. The invention includes the surprising finding that a high proportion of cells containing biochemical markers of dysplasia can be detected in superficial cervical epithelial cells (e.g. scrapings, lavage samples, swabs, etc.) from women with precancerous cervical dysplasia, avoiding the need for any cytological examination.
In one embodiment, the marker is a marker which portends apoptosis, such as markers of DNA fragmentation, caspase activity, membrane lipid distribution, subdiploid DNA content, etc. In another embodiment, the marker is a marker of angiogenesis, such as an endothelial cell-specific surface protein, secretory protein, growth factor, etc., angiogenic ephrin (Gibaldi M
J Clin Pharmacol
1998 Oct;38(10):898-903), angiogenic matrix metalloproteinases (Sang QX
Cell Res
1998 Sep;8(3):171-7), etc. Essentially the presence in superficial cervical epithelial cells of any biochemical marker, particularly markers of apoptosis and angiogenesis, the presence of which in subsuperficial cervical epithelium is correlated with dysplasia can be used in the invention.
In one embodiment of the invention, cells from a cervical scraping or lavage are suspended in a buffer solution containing digestive enzymes and mixed well. The resulting tissue and cells suspension is stained with a kit containing terminal deoxytransferase, fluorescent-labeled nucleotide and other reagents necessary for cell permeability, high labeling efficiency and low non-specific staining. The cells are scored for nuclear and cytoplasmic staining by either microscopic examination or laser flow cytometry. Dysplasia is diagnosed by the staining index after correction for non-epithelial cell staining.
REFERENCES:
Garzetti et al. Microinvasive Cervical Carcinoma and Cervical Intraepithelial Neoplasia; Biologic Significance and Clinical Implications of 72-kDa Metalloproteinase Immunostaining. May 1996, vol. 61, No
Grossnickle Ellen Beth
Razani Nooshin
Smith-McCune Karen
Bansal Geetha P.
Osman Richard Aron
The Regents of the University of California
LandOfFree
Biochemical methods for detecting cervical dysplasia and cancer does not yet have a rating. At this time, there are no reviews or comments for this patent.
If you have personal experience with Biochemical methods for detecting cervical dysplasia and cancer, we encourage you to share that experience with our LandOfFree.com community. Your opinion is very important and Biochemical methods for detecting cervical dysplasia and cancer will most certainly appreciate the feedback.
Profile ID: LFUS-PAI-O-2447667