DNA‐Cytometry of Progressive and Regressive Cervical Intraepithelial Neoplasia

Abstract

A retrospective analysis was performed on archival cervical smears from a group of 56 women with cervical intraepithelial neoplasia (CIN), who had received follow‐up by cytology only. Automated image cytometry of Feulgen‐stained DNA was used to determine the differences between progressive and regressive lesions. The first group of 30 smears was from women who had developed cancer after initial smears with dysplastic changes (progressive group). The second group of 26 smears with dysplastic changes had shown regression to normal (regressive group). The goal of the study was to determine if differences in cytometric features existed between the progressive and regressive groups. CIN categories I, II and III were represented in both groups, and measurements were pooled across diagnostic categories. Images of up to 700 intermediate cells were obtained from each slide, and cells were scanned exhaustively for the detection of diagnostic cells. Discriminant function analysis was performed for both intermediate and diagnostic cells. The most significant differences between the groups were found for diagnostic cells, with a cell classification accuracy of 82%. Intermediate cells could be classified with 60% accuracy. Cytometric features which afforded the best discrimination were characteristic of the chromatin organization in diagnostic cells (nuclear texture). Slide classification was performed by thresholding the number of cells which exhibited progression associated changes (PAC) in chromatin configuration, with an accuracy of 93 and 73% for diagnostic and intermediate cells, respectively. These results indicate that regardless of the extent of nuclear atypia as reflected in the CIN category, features of chromatin organization can potentially be used to predict the malignant or progressive potential of CIN lesions.

Epidemiologic modeling of cervical dysplasia with molecular and cytopathological markers

“…[8] Three categories of features (Table 1) were measured for each cell image: Morphological features characterize increases in nuclear size and severe distortions in nuclear shape that are associated with aneuploid progression of SIL. DNA content features estimate the absolute intensity, optical density levels of the nucleus (which are proportional to the DNA content of the cell), and the intensity distribution characteristics.…”

Elevated levels of tetraploid cervical cells in human papillomavirus-positive Papanicolaou smears diagnosed as atypical squamous cells of undetermined significance

“…47 The mechanism of aneuploidization in cervical carcinogenesis, while not fully elucidated, has also been hypothesized to follow a sequential pattern in which tetraploidy represents a transitional state between diploidy and aneuploidy. 23,26,27,29 An ongoing, continuing molecular epidemiologic study from our laboratory is focused on elucidating the mechanisms through which aneuploidization occurs during disease progression. Data from that continuing study indicate that tetraploidy is an unstable and transient chromosomal abnormality that is observed commonly during the middle and late disease stages from which the aneuploid lesions characteristic of cervical carcinoma subsequently develop.…”

“…For PCR, the MY09 and MY11 primers were used to amplify HPV DNA, and the GH20 and PC04 primers were used to amplify human ␤-globin DNA (Qiagen, La Jolla, CA). Amplification of the L1 gene can allow for the identification of at least 20 HPV types (types 6,11,16,18,26,31,33,35,39,40,42,45,51,52,53,54,55,56,57, and 59). The DNA samples were extracted and purified for PCR using a proteinase K-phenol/chloroform procedure.…”

“…A biomarker of an additional molecular event further along the pathway to cervical carcinogenesis may provide more specificity in the diagnosis and treatment of the disease. 22 Genetic abnormalities, such as numerical chromosomal aberrations (tetraploidy and aneuploidy), have been reported in women diagnosed with precancerous and cancerous cervical lesions [23][24][25][26][27][28][29][30] and are observed frequently in the presence of intermediate-risk and high-risk, HPV-type infections. [31][32][33] This correlation implies that these numerical chromosomal aberrations are the result of infection by certain types of HPV and may originate from the ability of the virus to abrogate key cellular tumor-suppressor proteins.…”

“…34 -38 It has been hypothesized that the numerical chromosomal aberrations observed during cervical carcinogenesis follow a sequential pattern in which the aneuploid cells characteristic of advanced cervical lesions develop through chromosomal loss from tetraploid intermediates. 26,29 Because it is believed that tetraploidy occurs subsequent to HPV infection and may represent a significant molecular event early in the development of cervical carcinoma, women who exhibit elevated levels of tetraploid cells may be at an increased risk of developing neoplastic lesions of the cervix. We examined 1000 cervical cells from each of 257 different women using multiple-probe fluorescence in situ hybridization (FISH) for chromosomes 3 and 17 to determine whether numerical chromosomal aberrations were present at elevated levels in equivocal ASCUS cervical smears from women who were infected with HPV.…”