Chemistry: molecular biology and microbiology – Treatment of micro-organisms or enzymes with electrical or... – Modification of viruses
Reexamination Certificate
1996-06-19
2001-07-10
Bansal, Geetha P. (Department: 1642)
Chemistry: molecular biology and microbiology
Treatment of micro-organisms or enzymes with electrical or...
Modification of viruses
C425S288000
Reexamination Certificate
active
06258576
ABSTRACT:
BACKGROUND OF THE INVENTION
I. Field of the Invention
The invention relates to optical methods and apparatus used for the diagnosis of cervical precancers.
II. Related Art
There has been a significant decline in the incidence of advanced cervical cancer over the last 40 years, primarily due to the development of organized programs that target early detection of its curable precursor, cervical Squamous Intraepithelial Lesion (SIL) (SILs consist of Cervical Intraepithelial Neoplasia (CIN) and Human Papilloma Viral (HPV) infection) [1]. Even though organized screening (Pap smear) and diagnostic (colposcopy) programs are currently in place, approximately 15,900 new cases of cervical cancer and 4,900 cervical cancer related deaths were reported in 1995, in the United States alone [2]. Currently, 24.5% of women with cervical cancer are under the age of 35 years, and the incidence continues to increase for women in this age group [1]. The continuing morbidity and mortality rate related to cervical cancer necessitates an improvement in the accuracy and efficacy of current detection modalities.
The Pap smear is the primary screening tool for the detection of cervical cancer and its precursor [3]. In a Pap test, a large number of cells obtained by scraping the cervical epithelium are smeared onto a slide which is then fixed and stained for cytologic examination. Each smear is then examined under a microscope for the presence of neoplastic cells [4]. The Pap smear's reported sensitivity and specificity range from 11-99% and 14-97%, respectively. Like many screening tests in an asymptomatic population, the Pap smear is unable to achieve a concurrently high sensitivity and high specificity [5]. The accuracy of the Pap smear is limited by both sampling and reading errors [6]. Approximately 60% of false-negative smears are attributed to insufficient sampling; the remaining 40% are due to reading errors. Because of the monotony and fatigue associated with reading Pap smears (50,000-300,000 cells per slide), the American Society of Cytology has proposed that a cyto-technologist should be limited to evaluating no more than 12,000 smears annually [7]. As a result, accurate Pap smear screening is labor intensive and requires highly trained professionals.
A patient with a Pap smear interpreted as indicating the presence of SIL is generally recommended for follow up with a diagnostic procedure called colposcopy [3]. During a colposcopic examination, the cervix is stained with acetic acid and viewed through a low power microscope to identify potential pre-cancerous sites; suspicious sites are biopsied and then histologically examined to confirm the presence, extent and severity of the SIL [8]. A patient who has high grade SIL (HG SIL) (which consists of CIN II and/or CIN III) is usually treated, whereas a patient diagnosed with low grade SIL (LG SIL) (which consists of HPV and/or CIN I) is generally followed further using colposcopy [3].
Colposcopic examination and tissue biopsy in expert hands maintains a high sensitivity (80-90%), at the expense of a significantly low specificity (50-60%) [9]. A poor specificity represents unnecessary biopsy of tissues which do not contain cervical pre-cancer. In spite of the poor specificity of this technique, extensive training is required to achieve this skill level. All biopsy specimens require histologic evaluation and, therefore, diagnosis is not immediate. The disconnection between colposcopic assessment and biopsy and definitive treatment is of particular concern in the management of economically disadvantaged patients who may not return for treatment, particularly since cervical cancer precursors are more prevalent in groups of lower socio-economic status [1].
Fluorescence spectroscopy is a technique that has the potential to improve the accuracy and efficacy of cervical pre-cancer screening and diagnosis. Fluorescence spectroscopy has the capability to quickly, non-invasively and quantitatively probe the biochemical and morphological changes that occur as tissue becomes neoplastic. The altered biochemical and morphological state of the neoplastic tissue is reflected in the spectral characteristics of the measured fluorescence. This spectral information can be correlated to tissue histopathology, the current “gold standard” to develop clinically effective screening and diagnostic algorithms. These mathematical algorithms can be implemented in software, thereby enabling automated, fast, non-invasive and accurate pre-cancer screening and diagnosis in the hands of non-experts.
Although a complete understanding of the quantitative information contained within a tissue fluorescence spectrum has not been achieved, many groups have applied fluorescence spectroscopy for real-time, non-invasive, automated characterization of tissue pathology. Characterization of tissue pathology using auto-fluorescence [10-23] as well as photosensitizer induced fluorescence [24-27] to discriminate between diseased and non-diseased human tissues in vitro and in vivo has been described in a variety of tissues.
Auto-fluorescence spectra of normal tissue, intraepithelial neoplasia and invasive carcinoma have been measured from several organ sites in vivo [13-17]. In vivo studies of the human colon at 370 nm excitation [13] indicated that a simple algorithm based on fluorescence intensity at two emission wavelengths can be used to differentiate normal colon and adenomatous polyps with a sensitivity and specificity of 100% and 97%, respectively. Shomacker et al. [14] conducted similar studies in vivo at 337 nm excitation and demonstrated that a multivariate linear regression algorithm based on laser induced fluorescence spectra can be used to discriminate between normal colon and colonic polyps with a similarly high sensitivity and specificity. Lam et al. developed a bronchoscope which illuminates tissue at 442 nm excitation and produces a false color image in near real-time which represents the ratio of fluorescence intensities at 520 nm (green) and 690 nm (red) [16,17]. In vivo studies demonstrated that the ratio of red to green auto-fluorescence is greater in normal bronchial tissues than in abnormal bronchial tissues [16]. In a trial with 53 patients, the sensitivity of fluorescence bronchoscopy was found to be 72%, as compared to 50% for conventional white light bronchoscopy [17].
Nonetheless, a reliable diagnostic method with improved diagnostic capability for use in vitro and in vivo is needed to allow faster, more effective patient management and potentially further reduce mortality.
SUMMARY OF THE INVENTION
The present invention demonstrates that fluorescence spectroscopy can be applied, both in vitro and in vivo, to the diagnosis of cervical tissue abnormalities including the clinical detection of cervical precancer.
In a first exemplary embodiment, there is provided a method of detecting tissue abnormality in a tissue sample comprising the steps of (i) providing a tissue sample; (ii) illuminating said sample with electromagnetic radiation wavelengths of about 337 nm, about 380 nm and about 460 nm to produce three fluorescence intensity spectra; (iii) detecting a plurality of emission wavelengths from said fluorescence intensity spectra; and (iv) establishing from said emission wavelengths a probability that said sample is abnormal. The illumination wavelengths are advantageously in the ranges of 317-357 nm, 360-400 nm and 440-480 nm. The method may further comprise preprocessing data at the emission wavelengths to reduce inter-sample and intra-sample variation. The establishing step may comprise normalizing the spectra relative to a maximum intensity within the spectra. Optionally, the establishing step does not comprise mean-scaling the spectra.
Emission wavelengths may be selected at about 410 nm, about 460 nm, about 510 nm and about 580 nm for an illumination o
Mahadevan-Jansen Anita
Mitchell Michele Follen
Ramanujam Nirmala
Richards-Kortum Rebecca
Bansal Geetha P.
Board of Regents , The University of Texas System
Fulbright & Jaworski LLP
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