Measurement of chemopreventive efficacy in skin biopsies

OBJECTIVE: To explore methods suitable for quantitative assessment of the efficacy of chemopreventive intervention. STUDY DESIGN: High-resolution imagery of nuclei from the suprabasal and basal cell layers of sun-damaged skin were recorded. There were 10 cases. A shave biopsy was taken from an area of clearly evident solar keratosis before and after treatment with 2-difluoromethyl-dlornithine (DFMO) and from the colateral forearm, treated with a placebo. A number of karyometric variables were computed and combined to derive marker features that provided a numeric measure of the degree of nuclear deviation from normal. RESULTS: DFMO treatment was effective overall in reducing the degree of nuclear abnormality seen in the biopsies; in 8 of the 10 cases there was a significant improvement. The placebo-treated arm did not show a statistically different abnormality from the untreated arm. CONCLUSION: Karyometric analysis can provide numeric measures that allow documentation of statistically significant regression of actinic keratotic lesions following treatment with DFMO.     

Karyometry in endometrial adenocarcinoma of different grades

OBJECTIVE: To characterize nuclei from adenocarcinoma of the endometrium of different grades in order to establish whether significantly different nuclear subpopulations exist. STUDY DESIGN: A total of 1,400 nuclei from 14 cases of adenocarcinoma of the endometrium of grades 1, 2 and 3 were recorded, and 600 nucleifrom normal, secretory phase glandular endometrial epithelium were used as a reference data set. Karyometric features were computed and a discriminant function derived to define a trend in feature values for nuclei from lesions of different grades. The nuclei from lesions of different grades were processed by a nonsupervised learning algorithm in an effort to detect and define subpopulations. RESULTS: Nuclei from grade 1 lesions represented a near-diploid stemline, whereas nuclei from grade 3 lesions formed an aneuploid set with a mode around 3N. The existence of three subpopulations with statistically different nuclear chromatin patterns could be shown for nuclei from each grade. In each grade these three subpopulations occupied the same relative position in feature space. For grade 1 lesions all three subpopulations were near diploid, for grade 3 lesions all three were approximately triploid. CONCLUSION: The nuclei in adenocarcinoma of the endometrium form a heterogeneous set, with subpopulations of distinctly different chromatin patterns and different ploidy. This should be taken into consideration in assessing the efficacy of chemopreventive intervention.     

Second order discriminant analysis in chemopreventive efficacy measurement

OBJECTIVE: To describe the use of second order discriminant analysis as a classification methodology along with the underlying assumptions and sampling requirements, with special emphasis on the use of this analysis in chemopreventive efficacy studies. STUDY DESIGN: The discriminant function score distributions derived in an analysis of 2 diagnostic groups may show such overlap that a statistically significant difference in mean values cannot be shown and, more important, that a useful case-based classification cannot be attained. By using the discriminant function score distributions from each case, it is frequently possible to derive a second order discriminant function based on case-specific characteristics, rather than characteristics of nuclei, thereby attaining improved case classification. RESULTS: Second order discriminant analysis has proven very useful in the documentation of case-level efficacy in chemopreventive trials. In a study of orally administered vitamin A, a first order discriminant analysis did not achieve a statistically significant difference in the score distributions for nuclei, but a second order discriminant analysis allowed a correct recognition of intervention effects in 85% of submitted cases. In a chemopreventive study of triamcinolone, a similarly inadequate discrimination based on discriminant function scores for nuclei resulted. After a second order discriminant analysis, a reduction in solar-actinic damage could be shown in 14/15, or 93%, of treated cases. CONCLUSION: Second order discriminant analysis can be highly effective when the discriminating information offered at the nuclear level is inadequate due to high dispersion and small differences in mean values of discriminant function scores for the diagnostic groups. Second order analysis utilizes case-specific characteristics of the discriminant function score distributions to document diagnostic group separation and/or efficacy of chemopreventive intervention by a reduction in case discriminant function scores.     

Progression curves for endometrial lesions

OBJECTIVE: To derive a numeric measure for the progression of endometrial lesions as a baseline study for an eventual assessment of chemopreventive intervention efficacy. STUDY DESIGN: Tissue sections from normal endometrium at the proliferative and secretory phase, simple hyperplasia, atypical hyperplasia from cases free of concomitant adenocarcinoma and adenocarcinoma of the endometrium were recorded at high spatial resolution. Six cases from each diagnostic category were chosen as "typical," and 60 epithelial nuclei were randomly selected for measurement for each case. Discriminant analyses were carried out to derive a direction of progressive change in feature space and to correct the progression curve for the presence of cells not expressing progressive change among the random sample of nuclei. RESULTS: A well-conditioned progression curve was derived based on the mean discriminant function scores for each diagnostic category and the mean nuclear abnormality of the nuclei in each category, as expressed by their deviation in feature values from normal reference nuclei. The lesion signatures showed a clear trend toward extension into the range of higher nuclear abnormalities with increasing progression. There was an indication that abnormal endometrial lesions may comprise cases with distinctly different degrees of nuclear abnormality. CONCLUSION: A numeric assessment of lesion progression for endometrial lesions, based on karyometric measurements, is possible. The data suggest that additional analysis may provide further characterizing information for individual lesions.     

Karyometric image analysis for intraepithelial and invasive cervical lesions

OBJECTIVE: To derive an objective, numeric measure for the progression of intraepithelial and invasive squamous cell cervical lesions. STUDY DESIGN: Thin-layer cervical cytology preparations from colposcopically confirmed normal cervix, low grade squamous intraepithelial lesions, high grade squamous intraepithelial lesions and carcinoma were identified from a cross-sectional study. Fifty-nine cases representing 4 diagnostic categories were selected, and 2,375 nuclei from epithelial cells representative of the diagnostic category were randomly selected for imaging and measurement from these cases. Additionally, 1,378 visually normal appearing intermediate cells from low and high grade squamous intraepithelial lesions, as well as from carcinoma cases, were identified for analysis. The nuclei were quantitatively characterized, and discriminant analyses were performed to derive a progression curve from normal cytology to carcinoma. RESULTS: The lesion signatures show a clear increase in nuclear abnormality with increasing progression. A progression curve was derived based on mean discriminant function scores for each diagnostic category and on the mean nuclear abnormality values for the nuclei in each category, as expressed by their deviation in feature values from normal reference nuclei. CONCLUSION: A numeric assessment of lesion progression for cervical precancerous and cancerous lesions based on karyometric measurements is possible and may provide an objective, precise characterization of each lesion as well as a basis for improved performance in automated cytology-based cervical cancer screening.     

Karyometric marker features in fine needle aspirates of invasive follicular carcinoma of the thyroid.

Karyometric measurements were performed on fine needle aspirates of clearly identifiable tumor areas and adjacent normal-appearing areas in the surgical specimens from ten patients with invasive follicular carcinoma of the thyroid. Similar measurements were performed on aspirates from nine patients free of thyroid disease (controls). A total of 95 karyometric features were evaluated for each nucleus. Analysis of variance of optical density values indicated (1) a similarity between tumor and normal-appearing nuclei from carcinoma cases, (2) a significant difference between those nuclei and control nuclei and (3) that most of the differences were due to the differences of tissue origin. Stepwise linear discriminant analysis selected ten features that produced a statistically highly significant separation of tumor nuclei from control nuclei. A similar analysis selected six features that produced a statistically highly significant discrimination of normal-appearing nuclei from control nuclei; the validity of those karyometric features as markers of malignancy in normal-appearing nuclei from tissues adjacent to invasive follicular carcinomas of the thyroid was demonstrated by analysis in further training and control sets of nuclei. This analysis in thyroid aspirates identified more marker features than did a previous similar analysis using tissue sections.     

Preneoplastic changes in ovarian tissues

OBJECTIVE: To test whether histologically normal epithelium within ovarian inclusion cysts and stroma exhibit changes in nuclear chromatin pattern that indicate the presence of occult ovarian lesions. STUDY DESIGN: Ovaries were collected from 10 low-risk women,from 7 high-risk women and from 3 women with ovarian cancer. Histologic sections were cut at 5 microm and hematoxylin and eosin stained. High-resolution images were recorded from the epithelium lining inclusion cysts and from the underlying stroma of ovaries from these 20 subjects. A total of 2860 epithelial nuclei and 3610 stromal nuclei were recorded. Karyometric features and nuclear abnormality were computed. Discriminant analyses and unsupervised learning algorithms defined deviations from normal that were designated "above threshold" and used to compute average nuclear abnormality of a second nuclear phenotype. RESULTS: Histologically normal epithelium from inclusion cysts of ovaries harboring a malignant lesion was shown to exhibit changes in the nuclear chromatin pattern that were statistically significant using quantitative image analysis procedures. Similar changes were seen in the inclusion cyst epithelia of high-risk ovaries. A subpopulation of cells representing a new phenotype was detected in the underlying stroma of women harboring a malignant ovarian lesion and in women at high risk of ovarian cancer. CONCLUSION: The karyometric changes observed in the epithelia lining inclusion cysts and in the underlying stroma of ovaries either with ovarian cancer or at high risk of ovarian cancer suggest the presence of preneoplastic changes in histologically normal tissue.     

Nuclear chromatin characteristics of breast solid pattern ductal carcinoma in situ.

OBJECTIVE: To characterize nuclei from breast solid pattern ductal carcinoma in situ (DCIS) by their karyometric features and to search for the presence of statistically significantly different subsets of nuclei. STUDY DESIGN: One hundred nuclei from each of 6 normal, 13 solid DCIS, (9 low and intermediate grade and 4 high grade DCIS) histopathologic samples of breast tissue were digitally recorded. Karyometric features were computed and subjected to a nonsupervised learning algorithm (P-index) to identify significantly different subgroups. RESULTS: Nuclei in low grade lesions displayed a diploid/near diploid pattern, while the majority of intermediate grade lesions fell into a range beyond 5N. The high grade lesions showed substantial genomic instability and represented three statistically different subsets or phenotypes. CONCLUSION: There is a progression of nuclear abnormality from low grade to high grade DCIS. The nuclei from high grade DCIS form a heterogeneous set that represents three phenotypes. One of these phenotypes shows a nuclear chromatin pattern that more closely resembles poorly differentiated, infiltrating disease. The observation of such a phenotype may have prognostic implications.     

Karyometry of nuclei from actinic keratosis and squamous cell cancer of the skin

OBJECTIVE: To use karyometric analysis methods to compare actinic keratoses (AKs) to squamous cell carcinomas (SCCs) to determine if SCCs showed a logical progression beyond that seen in AKs and to explore variability within and between lesion types to better understand distinctions between the 2. STUDY DESIGN: Biopsies from 31 subjects with AKs were obtained from upper inner arm skin, forearm skin and AK lesions. Biopsies from 23 different subjects in a related subproject provided SCC biopsies for comparison. RESULTS: Karyometric measures of nuclear abnormality and sun damage were derived. Mean actinic damage levels progressed logically from inner arm to sun-exposed skin, to AK, to SCC. Considerable heterogeneity existed at the case level. Unsupervised learning methods revealed 2 distinct clusters of progressed lesions with different nuclear signatures, reflecting differing levels of actinic damage. Number of AKs and SCCs and invasiveness and differentiation of SCCs were distributed across both clusters in roughly equivalent proportions. CONCLUSION: Karyometric methods, shown previously to be capable of sensitively detecting subtle nuclear changes, revealed the possibility of 2 progression pathways, each containing AKs and SCCs. This finding may have prognostic implications.     

Karyometric analysis of actinic damage in unexposed and sun-exposed skin and in actinic keratoses in untreated individuals

OBJECTIVE: To assess the ability of karyometric analysis to demonstrate progression of actinic damage as a function of sun exposure in individuals with actinic keratoses (AKs) and to evaluate the stability of that assessment over a 3-month period. STUDY DESIGN: Biopsies from subjects with AKs were obtained from unexposed skin, sun-exposed skin and AK lesions. Subjects used an SPF 50 sunscreen, and 3 months later additional biopsies were taken from sun-exposed and AK sites. A total of 13,300 nuclei were recorded from 31 subjects. RESULTS: Measures of nuclear abnormality (NA) and effects of sun damage based on discriminant function (DF) scores were derived. Actinic damage levels varied significantly across biopsy site, demonstrating the method's sensitivity. Accrual of actinic damage was demonstrated in sun-exposed skin and AK lesions when all nuclei were examined over 3 months but only for sun-exposed skin when the worst-damaged nuclei were examined. This suggests a ceiling effect of nuclear damage in the progression to abnormality. Within-subject variability was similar for both NA and DF when all nuclei were considered. Among the worst-damaged nuclei (as defined by high DF), DF showed lower within-case variability than NA, perhaps due to a reduction in nuclear heterogeneity. CONCLUSION: Karyometry's ability to detect subtle levels of actinic damage in nuclear chromatin patterns may make it useful in screening agents for possible use in cancer chemoprevention.     

Karyometric marker features in fine needle aspirates of follicular adenoma of the thyroid

Karyometric measurements were performed on fine needle aspirates of clearly identifiable adenomatous areas and adjacent normal-appearing areas in the surgical specimens from ten patients with follicular adenomas of the thyroid. Similar measurements were made on aspirates from nine patients free of thyroid disease. A total of 95 karyometric features were evaluated for each nucleus. Analysis of variance of optical density values did not show a significant difference between the three types of nuclei. Discriminant analysis selected seven karyometric features that produced a statistically highly significant separation of adenoma nuclei from control nuclei. A similar analysis selected six features that produced a statistically highly significant discrimination of normal-appearing nuclei from control nuclei. The validity of these markers for distinguishing control nuclei from adenoma nuclei and normal-appearing nuclei adjacent to adenomas was demonstrated by analysis in further training and test sets. These findings parallel those previously demonstrated for invasive and microinvasive follicular carcinomas of the thyroid.     

Karyometry in Barrett's esophagus

OBJECTIVE: To derive a progression curve for lesions in Barrett's esophagus based on karyometric features. STUDY DESIGN: High-resolution imagery of 900 nuclei from normal gastric tissue, Barrett's metaplasia, Barrett's high grade dysplasia and adenocarcinoma of the esophagus was recorded. Karyometric features were computed, and nuclear signatures and lesion signatures for these lesions were derived. A progression curve was defined. RESULTS: Esophageal lesions were distinctly different from the normal gastric fundus tissue, with nuclei from Barrett's metaplasia deviating from normal almost as much as nuclei from high grade dysplasia and adenocarcinoma. There was considerable case-to-case variability and overlap between lesions histologically assigned to different diagnostic categories. CONCLUSION: The karyometric data suggest that Barrett's metaplasia is a more developed lesion than previously assumed.     

Quantitative analysis of prostatic intraepithelial neoplasia on tissue sections.

The changes in nuclear morphology (karyometry) and DNA content in prostatic intraepithelial neoplasia (PIN) were analyzed on tissue sections. The cases of PIN were subdivided into PIN 1 and PIN 2 based on the degree of proliferation and the anaplasia of the secretory cells lining the ducts and acini. Cases of nodular hyperplasia (NH) and adenocarcinoma were also studied for comparative purposes. Karyometric analysis showed a progression of most values from NH to PIN to carcinoma. The DNA analysis showed a decrease in the frequency of nuclei in the diploid range and an increase in the percentage of nuclei in the other ploidy regions (especially between 2c and 4c and in the tetraploid range) from NH to PIN to carcinoma. Forward stepwise discriminant analysis showed similarities between NH and PIN 1 and between PIN 2 and carcinoma. These findings suggest that the evolution towards adenocarcinoma is characterized by progressive morphologic derangements of the nuclei and by the transformation of the diploid DNA content into a nondiploid one, with the changes taking place at the level of PIN 2.     

Karyometry in rectal mucosa of patients with previous colorectal adenomas

OBJECTIVE: To determine whether karyometric measurements taken in biopsies from histologically normal-appearing rectal mucosa could serve as a biomarker for the risk of recurrence of polyps. MATERIALS AND METHODS: Biopsies were taken from the rectal mucosa of cases with a prior history of colonic polyps at the baseline of the study. In 57 cases recurrent polyps occurred (R cases); in 72 cases no recurrent disease was found at the end of the study (NR cases). From each biopsy 100 nuclei were recorded at high resolution. After segmentation, feature extraction and selection of a discriminating subset of features, a number of discriminant functions were derived. Also, measures of nuclear abnormality were computed. RESULTS: The differences in karyometricfeature values for nuclei from biopsies of cases with recurrent or nonrecurrent disease were very small and not notably expressed in the majority of nuclei. It was possible by focusing on nuclei showing clear deviations from normal to derive a discriminant function that exhibited a shift for the NR and R data sets. The distributions of discriminant function scores were then subjected to a second-order discriminant analysis to separate cases according to recurrence status. This function showed a statistically highly significant correlation with recurrence. At one extreme of its score distribution were 11 of 57 cases that had a recurrence, and at the other extreme were 8-10 of 72 cases that had no recurrence. The distributions of nuclear abnormality values for these subsets of cases were drastically different, with an average value of 1.72 for the group that may be at high risk for another recurrence and 1.02 for the group possibly at low risk. All cases with a prior history of colonic polyps showed a nuclear abnormality deviating from normal. CONCLUSION: Measurement of a sample of 100 nuclei from the rectal mucosa will suggest, for approximately 10% of cases, that a high risk for recurrence of adenomatous polyps exists and, for a slightly lower proportion, confirm that the nuclei deviate only slightly from those from individuals with no history of colonic polyps. For the majority of cases with a prior history of adenoma, the nuclei in the biopsy show a notable deviation from normal, but the deviation is practically the same for cases that had a recurrence and those that did not. However, a tentative discriminant function (DF I,3) derived from the characteristics of the extreme cases correctly classified approximately 64% of nonrecurrent and 83% of recurrent cases using a Bayesian decision boundary.     

Karyometric marker features in tissue adjacent to invasive cervical carcinomas

A companion study showed the existence of statistically significant changes in the value of karyometric "marker features" in the nuclei of histologically normal-appearing ectocervical epithelium adjacent to carcinomas in situ. In this second part of the study, the results obtained in patients with invasive cervical carcinomas were analyzed. Formalin-fixed, paraffin-embedded tissue sections were Feulgen stained and analyzed with a microTICAS video microphotometer. The results, demonstrated for the first time in histologic material, indicate that the marker features are clearly expressed in a majority of nuclei observed in the normal-appearing tissue adjacent to invasive lesions. This effect was statistically significant. The best marker features selected by the discriminant analysis were nuclear roundness, nuclear perimeter length, total optical density and a run length texture measure. These findings may reflect a subtle transformation of the apparently normal cervical epithelium adjacent to an invasive cervical carcinoma.     

Image analysis of mesothelioma. I. differentiation of mesothelioma from adenocarcinoma of the lung

OBJECTIVE: To explore the usefulness of nuclear micromorphometric analysis for the differentiation between epithelial mesothelioma and metastatic adenocarcinoma in the chest wall. STUDY DESIGN: High-resolution images of 2,100 nuclei from 27 cases of epithelial mesothelioma and 15 cases of adenocarcinoma of the lung were recorded. Stepwise discriminant analysis and a nonparametric classifier were applied to derive estimates for a case diagnosis correct classification rate. RESULTS: Nuclei from epithelial mesothelioma and adenocarcinoma of the lung showed statistically significantly different properties, but there was a region of overlap in feature space such that approximately 15-20% of cases could not be correctly classified. The lesion signatures derived from the mesothelioma cases with discriminant function scores that might result in case misclassification and the cases of adenocarcinoma of the lung spanned a similar range of degree of nuclear abnormality. However, the distribution of nuclear abnormality values for the mesothelioma cases has a mode at 0.87 SD from normal, whereas the distribution seen in lung adenocarcinoma cases had a mode at about 3.7 SD. CONCLUSION: Cases of epithelial mesothelioma and adenocarcinoma of the lung have nuclei with a wide range of deviation from normal in the spatial and statistical distribution of their nuclear chromatin. For approximately 80% of cases, correct case classification can be provided by nuclear micromorphometric analysis. Cases of epithelial mesothelioma with highly abnormal nuclei overlap in feature space with nuclei from adenocarcinoma of the lung. However, it is possible that characterization by a lesion signature may allow correct assignment for those cases.     

Malignancy-associated changes in breast tissue detected by image cytometry.

In several tissues, nuclear differences have been described in normal-appearing cells from patients with invasive carcinomas compared to cases without invasive carcinoma, a phenomenon known as malignancy-associated changes (MACs). The aim of this study was to determine the presence of malignancy-associated changes in breast tissue. Image cytometry was performed on Feulgen stained tissue sections of patients with usual ductal hyperplasia with (n = 30) or without (n = 41) adjacent invasive breast carcinoma. Nuclear features of normal-appearing cells as well as of usual ductal hyperplastic cells were separately compared between the two groups. Many features of normal-appearing epithelial cells were significantly different between cases with and without invasive cancer. Significant differences were also found by measuring ductal hyperplastic nuclei instead of normal-appearing nuclei. Cases with or without cancer could be distinguished with a classification accuracy of 80% by discriminant analysis using 2 nuclear features derived from ductal hyperplastic cells. In conclusion, image cytometry on breast tissue sections shows that malignancy-associated changes can be found in normal as well as in usual ductal hyperplastic breast cells. This could be clinically relevant for the detection of occult breast cancer, for the prediction of risk in these lesions, and to monitor the effect of chemopreventive agents.     

Quantitative cytomorphologic analyses of Papanicolaou-stained smears from oral lichen planus

OBJECTIVE: To apply quantitative techniques to cytologic smears in order to improve the diagnostic sensitivity of cytology in the detection of malignant change in the oral cavity. STUDY DESIGN: Eighty-two patients with lesions of oral lichen planus were investigated. A total of 247 Papanicolaou-stained buccal smears underwent nuclear and cytoplasmic area measurements using a TV image analyzer. RESULTS: The cytomorphologic results of this study suggest that lesions of oral lichen planus contain smaller cells and nuclei than those observed in clinically normal oral mucosal smears. There was a statistically significant reduction in nuclear area (P < .001) and cytoplasmic area (P < .05) for the oral lichen planus smears. CONCLUSION: The TV image analysis system could provide a valuable method of quantifying cell changes in oral smears collected from oral mucosa as a method of monitoring lesions of oral lichen planus.