Detection of epithelial dysplasia

A system to detect epithelial dysplasia is disclosed by examining cells obtained by a non-lacerational trans-epithelial device. The system selects suspect cells based on abnormal morphology, cytometry and/or using molecular diagnostic techniques. In a preferred embodiment, the results of the computer analysis are displayed as a histogram and the images of the suspect cells are reviewed for DNA ploidy by a pathologist on a cell by cell basis.

DETAILED DESCRIPTION OF THE INVENTION AND THE PREFERRED EMBODIMENT In the preferred embodiment, the presence of abnormal cellular morphology, abnormal keratinization and/or abnormal DNA ploidy, as detected by obtaining a non-lacerational trans-epithelial cellular sample, are combined with methods that demonstrate molecular alterations of cells from that trans-epithelial cellular sample to increase the sensitivity of 1) detection of epithelial lesions that are dysplastic or cancerous and 2) detection of epithelial lesions that will progress to carcinoma. An advantage of the subject invention over the prior art is greater sensitivity as an indicator of dysplasia and of a developing carcinoma, even preceding morphological tissue alterations. The trans-epithelial sample is preferably obtained using the device disclosed in the '186 application, the disclosure of which is fully incorporated herein by reference. Epithelial lesions that display “atypical” cellular changes in a trans-epithelial cellular sample may or may not be of significance since some lesions represent carcinoma, others represent premalignancy and yet others represent benign lesions which may ultimately become malignant. By combining a DNA ploidy analysis of the trans-epithelial cellular specimen with a molecular diagnostics determination, the present invention can be utilized as a method of increasing the sensitivity for identifying those atypical epithelial lesions which will progress to carcinoma as well as identifying those which will not. As an important feature of this invention, the trans-epithelial sample of epithelial tissue is examined for abnormalities in cellular morphology, DNA concentration, and keratinization as disclosed in the pending '218 and '219 applications and/or examined for other abnormalities in cellular morphology using computer assistance as disclosed in those applications. Atypical cells are selected for by the computer and a DNA ploidy determination of the suspect cells is then conducted by a pathologist. Additionally, the sample may be analyzed with molecular diagnostic techniques including, but not limited to, fluorescence and non-fluroescence in situ hybridization, loss of heterozygosity, clonal genetic alterations, PCR, p53 expression and the expression pattern of CD44 variant 6 protein by immunohistochemistry, monoclonal antibodies reactivity patterns, glutathione S-transferase activity, quantitative assessment of nucleolar organizer regions and cell cycle and proliferation markers such as the centromere-associated protein. Molecular diagnostic as well as DNA ploidy determination techniques that have been utilized to date have been performed on cellular specimens obtained from either invasive, lacerational biopsies or from scrapings of superficial cells using cytologic instruments. An advantage of this invention is the application of a DNA ploidy analysis and molecular diagnostic techniques to cellular samples obtained with a noninvasive apparatus such as that disclosed in the U.S. Pat. No. 6,258,044, which samples cells from all levels of an epithelial lesion. Another advantage of this invention is the increased sensitivity compared to all existing methods by themselves, including histopathology, cytology, and molecular diagnostic techniques of identifying dysplasia in epithelial tissue and the detection of epithelial lesions that may progress to carcinoma as well as those which may not. The molecular diagnostic techniques can be applied before or after the trans-epithelial sample of epithelial tissue is examined for abnormalities in cellular morphology, abnormalities in keratinization or abnormalities in DNA ploidy as disclosed in the pending '218 and '219 applications and/or examined for other abnormalities in cellular morphology using computer assistance as disclosed in those applications. Furthermore, the DNA ploidy determination may be made either independently or in conjunction with the molecular diagnosis, but such DNA ploidy examination is always made in conjunction with the methods and systems of the '218 and '219 applications. Because most of the interpretations of DNA measurements are population-based, the results of the computer analysis may be displayed as a DNA histogram. In a further embodiment, a histogram is plotted based on the DNA ploidy of the cell population. “Clean” cells, exhibiting normal nuclear to cytoplasmic ratios and morphology, are chosen from the population. This allows for the indication of atypical cells relative to the “normal” looking cells found within the same population and serves to eliminate the reduced sensitivity associated with using a blind control. Additionally, errors associated with estimating the DNA ploidy of a cell population are eliminated due to the fact that the final DNA ploidy determination is conducted by a pathologist on a cell by cell basis. Dysplasia is characterized as being either high-risk (aneuploid), intermediate-risk (tetraploid) or a low risk (diploid) lesion. As the pathologist reviews the sample, an indicator on the histogram serves to represent the relative DNA ploidy determination found for an individual cell of interest. In a preferred embodiment, a light indicator on the histogram alerts the pathologist as to the DNA ploidy of the selected cell of interest. 
 Results FIGS. 1 - 3 present data from superficial, intermediate and basal cell layers of the oral cavity. Each quadrant contains a suspect cell found within the population under review and includes a nuclear to cytoplasmic ratio displayed in the bottom left hand corner. FIGS. 1 and 2 display atypical cells warranting further investigation of the respective patient. Both Figs. show an increase in the nuclear staining, an increase in the nuclear cytoplasmic ratio, and nuclear crowding with a loss of polarity. In FIG. 1 , quadrants 10 , 15 , 20 , 25 , 30 , 35 , 40 , 45 , 50 , and 55 show an increase in nuclear staining. Of special concern, quadrant 10 indicates that the cell of interest has a high nuclear to cytoplasmic ratio (of 1:9). This is observed by an increase in density as the nucleus absorbs a larger portion of the cytometric dye. The ability to examine individual cells of interest gives the pathologist a greater degree of accuracy. Further investigation may include additional harvesting of cells from the region of interest. Similarly, FIG. 2 displays cells of a second patient also warranting further investigation by a pathologist. Quadrants 60 and 65 indicate a relatively high nuclear to cytoplasmic ratio of 1 to 13 and 1 to 17, respectively. Of additional concern, quadrants 125 and 130 contain naked nuclei surrounded by a bloody background. By examining the actual cell the pathologist is able to determine that the low nuclear to cytoplasmic ratio is attributed to a cell which is no longer intact. FIG. 3 . shows cells positive for dysplasia or carcinoma. As indicated by the display in the bottom left hand corner of quadrants 150 , 155 and 160 , there is a dramatic increase in the nuclear to cytoplasmic ratio. Upon further observance by a pathologist, it is noted the cells have an irregular shape. The computer based retrieval of cells containing a combination of irregular shape and nuclear DNA concentration allows the pathologist to quickly focus on cells of interest. Again, regions of interest may be revisited and additional cells harvested by the pathologist. The final interpretation of the image analysis histogram may be conducted in conjunction with the patient's history, biopsy findings, or any other pertinent test results. For example: all the image results may then be integrated into the corresponding biopsy report and discrepancies between the two addressed. Having described this invention with regard to specific embodiments, it is to be understood that the description is not meant as a limitation since further embodiments, modifications and variations may be apparent or may suggest themselves to those skilled in the art. It is intended that the present application cover all such embodiments, modifications and variations. 1 Diagram 1 Taking a trans-epithelial sample of epithelial tissue ↓ Examining said trans-epithelial sample of epithelial tissue for abnormalities in cellular morphology and abnormalities in keratinization and/or examining said sample of epithelial tissue for abnormalities using computer-assisted analysis, including but not limited to the machines and/or techniques of the ‘218 and/or ‘219 applications. ↓ Analyzing the sample with a molecular diagnostic technique, said technique including but not limited to, fluorescence in situ hybridization, loss of heterozygosity, clonal genetic alterations, PCR, p53 expression and the expression pattern of CD44 variant 6 protein by immunohistochemistry, monoclonal antibodies reactivity patterns, glutathione S-transferase activity, measuring the number of nucleolar organizer regions and cell-cycle and proliferation markers such as the centromere-associated protein. 2 Diagram 2 Taking a trans-epithelial sample of epithelial tissue ↓ Examining said trans-epithelial sample of epithelial tissue for abnormalities in cellular morphology, and DNA concentration and/or examining said sample of epithelial tissue for abnormalities using computer-assisted analysis, including but not limited to the machines and/or techniques of the ‘218 and/or ‘219 applications. ↓ Analyzing the sample for a DNA ploidy analysis, said DNA ploidy determination being conducted by a pathologist. 3 Diagram 3 Taking a trans-epithelial sample of epithelial tissue ↓ Examining said trans-epithelial sample of epithelial tissue for abnormalities in cellular morphology, keratinization and DNA concentration and/or examining said sample of epithelial tissue for abnormalities using computer-assisted analysis, including but not limited to the machines and/or techniques of the ‘218 and/or ‘219 applications. ↓ Analyzing the sample with a molecular diagnostic technique and/or for a DNA ploidy analysis, said DNA ploidy determination being conducted by a pathologist and said molecular diagnostic technique including but not limited to, fluorescence in situ hybridization, loss of heterozygosity, clonal genetic alterations, PCR, p53 expression and the expression pattern of CD44 variant 6 protein by immunohistochemistry, monoclonal antibodies reactivity patterns, glutathione S-transferase activity, measuring the number of nucleolar organizer regions and cell-cycle and proliferation markers such as the centromere-associated protein.