Hubert G. Bartels

Analysis of the capillary architecture in the precursors of prostate cancer : Recent findings and new concepts

OBJECTIVE To report on recent findings and new concepts in the remodeling of the capillary architecture in the precursors of prostate cancer. METHODS Immunohistochemical methods have been adopted in prostate cancer and in its precursors (prostatic intra-epithelial neoplasia) to investigate capillary pattern changes-which were mainly analyzed as capillary frequency- and the degree of endothelial cell proliferation. Several features related to the capillary architecture have been considered. Manual, semiautomatic, and automatic (machine vision) types of evaluation have been used to quantify the features. RESULTS The data available indicate that: (1) Going from normal prostate through prostatic intra-epithelial neoplasia up to invasive adenocarcinoma, an increasing proportion of capillaries becomes shorter, with open lumen and undulated external contour and with greater proliferation of the endothelial cells and greater expression of type IV collagenase. The highest proportion of touching capillaries is seen in normal prostate, while the lowest is found in invasive adenocarcinoma, being intermediate in prostatic intra-epithelial neoplasia. (2) When total androgen ablation is induced, there is no proliferation of the endothelium, whereas the capillaries are reduced in frequency and represented by small vessels lined by flat endothelial cells and with an open lumen. (3) Automation in the evaluation of the capillary architecture is feasible with a machine vision system. CONCLUSIONS The progression in prostate carcinogenesis is associated with changes in the capillary architecture. There are some preliminary data indicating that total androgen ablation can inhibit the angiogenesis in precursors of prostate cancer.

Data representation and reduction for chromatin texture in nuclei from premalignant prostatic, esophageal, and colonic lesions

BACKGROUND To identify nuclei and lesions with great specificity, a large set of karyometric features is arranged in the form of a linear profile, called a nuclear signature. The karyometric feature values are normalized as z-values. Their ordering along the profile axis is arbitrary but consistent. The profile of the nuclear signature is distinctive; it can be characterized by a new set of variables called contour features. A number of data reduction methods are introduced and their performance is compared with that of the karyometric features in the classification of prostatic, colonic, and esophageal lesions. METHODS Contour characteristics were reduced to descriptive statistics of the set of z-values in the nuclear signature and to sequence information. The contour features derived were (1) relative frequencies of occurrence of z-values and of their differences and (2) co-occurrence statistics, run lengths of z-values, and statistics of higher-order dependencies. Performance was evaluated by comparing classification scores of diagnostic groups. RESULTS Rates for correct classification by karyometric features alone and contour features alone indicate equivalent performance. Classification by a combined set of features led to an increase in correct classification. CONCLUSIONS Image analysis and subsequent data reduction of nuclear signatures of contour features is a novel method, providing quantitative information that may lead to an effective identification of nuclei and lesions.

Karyometry of the Colonic Mucosa

Objective: The study summarizes results of karyometric measurements in epithelial cells of the colorectal mucosa to document evidence of a field effect of preneoplastic development among patients with colorectal adenocarcinoma or adenoma. Methods: Karyometric analyses were done on high-resolution images of histologic sections from 48 patients with colorectal adenocarcinomas and 44 patients with adenomas and on images from matching normal-appearing mucosa directly adjacent to such lesions, at a 1-cm and 10-cm distance from the lesions or from the rectal mucosa of adenoma patients, as well as from 24 healthy normal controls with no family history of colonic disease. Results: The nuclei recorded in the histologically normal-appearing mucosa of patients with either colorectal adenoma or adenocarcinoma exhibited differences in karyometric features in comparison with nuclei recorded in rectal mucosa from patients who were free of a colonic lesion. These differences were expressed to the same extent in tissue adjacent to the lesions and in normal-appearing tissue as distant as the rectum. Conclusions: The nuclear chromatin pattern may serve as an integrating biomarker for a preneoplastic development. The field effect might provide an end point in chemopreventive intervention trials. (Cancer Epidemiol Biomarkers Prev 2007;16(12):2704–16)

Tissue Architecture Analysis in Prostate Cancer and Its Precursors: An Innovative Approach to Computerized Histometry

Aims: It is the aim of these studies to derive a numerically defined progression index for prostatic intraepithelial neoplasia (PIN) lesions. Methods: Histometric and karyometric features were automatically extracted from images of histopathologic sections by a machine vision system. Results: Both histometric and karyometric measures lend themselves to the defining of a progression index. Karyometric features were found to be more sensitive. They allow the detection of very early change. Conclusions: It is possible to measure progression of PIN lesions with precision. The methodology would lend itself for measurement of regression due to chemopreventive intervention.

Quantitative Study of Breast Cancer Progression: Different Pathways for Various In Situ Cancers

The chromatin pattern in nuclei from breast ductal proliferative lesions was quantitatively evaluated with the objective of deriving measures of tumor progression. A total of 110 cases were analyzed. There were 38 cases of normal tissue or benign proliferative lesions, 41 cases of ductal carcinoma in situ (DCIS), and 31 cases of microinfiltrating DCIS and of infiltrating cancer. A total of 9424 nuclei were analyzed. High-resolution images were digitally recorded. For each nucleus, 93 karyometric features descriptive of the spatial and statistical distribution of the nuclear chromatin were computed. Data analysis included establishing a profile of relative deviations of each feature from “normal,” called the nuclear signature, and of lesion signatures as well as of trends of lesion progression. Two trends of evolution could be discerned: one from normal to hyperplasia, atypical hyperplasia, and comedo DCIS as representative of high-grade lesions; and the other from normal to hyperplasia to cribriform DCIS, solid DCIS, and infiltrating cancer, representing lower grade lesions. The nuclei in microinfiltrating foci are distinctly different from nuclei in high-grade comedo DCIS. The nuclei in microinfiltrating foci have a statistically significantly lower nuclear abnormality. They may represent outgrowing clones.

Scene segmentation in a machine vision system for histopathology

Algorithms and procedures employed to attain reliable and exhaustive segmentation in histopathologic imagery of colon and prostate sections are detailed. The algorithms are controlled and selectively called by a scene segmentation expert system as part of a machine vision system for the diagnostic interpretation of histopathologic sections. At this time, effective segmentation of scenes of glandular tissues is produced, with the system being conservative in the identification of glands; for the segmentation of overlapping glandular nuclei an overall success rate of approximately 90% has been achieved.

Nuclear Morphometry Identifies a Distinct Aggressive Cellular Phenotype in Cutaneous Squamous Cell Carcinoma

By identifying aggressive cutaneous squamous cell carcinoma (cSCC) in patients who are at high risk for recurrences or second primaries after resection, intensive surveillance and therapy may decrease morbidity and mortality. We investigated the role of nuclear morphometry (karyometry) in differentiating between aggressive and nonaggressive cSCC. We retrospectively analyzed cSCC lesions from 40 male patients. Twenty-two patients had evidence of aggressive cSCC (local/regional recurrence or a second primary cSCC), and 18 patients were identified with similar ages and sites of disease as control patients with nonaggressive cSCC (no evidence of recurrence, metastasis, or second primary). We carried out karyometric analysis to identify nuclear features that discriminate between aggressive and nonaggressive cSCC nuclei. We used statistically significant differences (Kruskal–Wallis test, P < 0.0001) to compose a quantitative aggressive classification score (proportion of aggressive nuclei from 0% to 100%). For comparisons, we used Fishers exact test or Students t test. The mean age was 79 ± 7 years for aggressive cSCC and 80 ± 9 years for nonaggressive cSCC (P = 0.66). We analyzed a mean of 96 nuclei in each group. The mean classification score for aggressive cSCC was significantly higher (69% ± 6%) than for nonaggressive cSCC (28% ± 5%, P = 0.00002). Overall, the classification score accurately categorized 80% of our patients (P = 0.0004). In most patients, karyometry differentiated between aggressive and nonaggressive cSCC. We found that classification scores, which provide information on individual lesions, could be used for risk stratification. Cancer Prev Res; 4(11); 1770–7. ©2011 AACR.

Automatic segmentation of cell nuclei in bladder and skin tissue for karyometric analysis

Objective: To automatically segment cell nuclei in histology images of bladder and skin tissue for karyometric analysis. Materials/Methods: The four main steps in the program were as follows: 1) median filtering and thresholding, 2) segmentation, 3) categorizing, and 4) cusp correction. This robust segmentation technique used properties of the image histogram to optimally select a threshold and create closed four-way chain code nuclei segmentations. Each cell nucleus segmentation was treated as an individual object with properties of segmentation quality. A segmentation was placed in one of the following three categories based on its properties: throw away, salvageable, or good. Erosion/dilation and rethresholding were performed on salvageable nuclei to correct cusps. Results: Ten bladder histology images were segmented both by hand and using this automatic segmention algorithm. The automatic segmentation resulted in a sensitivity of 76.4%. The average difference between hand and automatic segmentations over 42 nuclei, calculated for each of the 95 features used in karyometric analysis, ranged between 0 and 48.3%, with an average of 2.8%. The same procedure was performed on 10 skin histology images with a sensitivity of 83.0%. Average differences over 44 nuclei ranged between 0 and 200%, with an average of 10.0%. Conclusion: The close agreement in karyometric features with hand segmentation shows that automated segmentation can be used for analysis of bladder and skin histology images. Average differences between hand and automatic segmentations were smaller in bladder histology images because these images contained less contrast, and therefore the range of the karyometric feature values was smaller.

Phase IIB Randomized Study of Topical Difluoromethylornithine and Topical Diclofenac on Sun-Damaged Skin of the Forearm

Prevention of nonmelanoma skin cancers remains a health priority due to high costs associated with this disease. Diclofenac and difluoromethylornithine (DFMO) have demonstrated chemopreventive efficacy for cutaneous squamous cell carcinomas. We designed a randomized study of the combination of DFMO and diclofenac in the treatment of sun-damaged skin. Individuals with visible cutaneous sun damage were eligible. Subjects were randomized to one of the three groups: topical DFMO applied twice daily, topical diclofenac applied daily, or DFMO plus diclofenac. The treatment was limited to an area on the left forearm, and the duration of use was 90 days. We hypothesized that combination therapy would have increased efficacy compared with single-agent therapy. The primary outcome was change in karyometric average nuclear abnormality (ANA) in the treated skin. Individuals assessing the biomarkers were blinded regarding the treatment for each subject. A total of 156 subjects were randomized; 144 had baseline and end-of-study biopsies, and 136 subjects completed the study. The ANA unexpectedly increased for all groups, with higher values correlating with clinical cutaneous inflammation. Nearly all of the adverse events were local cutaneous effects. One subject had cutaneous toxicity that required treatment discontinuation. Significantly more adverse events were seen in the groups taking diclofenac. Overall, the study indicated that the addition of topical DFMO to topical diclofenac did not enhance its activity. Both agents caused inflammation on a cellular and clinical level, which may have confounded the measurement of chemopreventive effects. More significant effects may be observed in subjects with greater baseline cutaneous damage. Cancer Prev Res; 9(2); 128–34. ©2015 AACR. See related article by Tsai and Hawk, p. 125

Global acetylation and methylation changes predict papillary urothelial neoplasia of low malignant potential recurrence: a quantitative analysis.

Papillary urothelial neoplasia of low malignant potential (PUNLMP) recurs in approximately 35% of patients. Conventional histopathological assessment does not distinguish non-recurrent from recurrent PUNLMP. The aim of this study is to explore the differences in global histone acetylation and global DNA methylation between non-recurrent and recurrent PUNLMP. Acetylated histone H3 lysine 9 (AcH3K9) and 5-methylcytosine (5MeC) were investigated by immunohistochemistry (IHC) in 20 PUNLMP cases (10 non-recurrent and 10 recurrent), in 5 cases of normal urothelium (NU) and in 5 cases of muscle invasive pT2 urothelial carcinoma (UC). The total optical density of the nuclear staining was measured photometrically in at least 40 nuclei separately for the basal, intermediate and luminal positions in each case. Concerning the total optical density values for both acetylation and methylation, a decrease in staining is observed from non-recurrent PUNLMP to recurrent PUNLMP, at all nuclear locations. For acetylation the mean value in non-recurrent PUNLMP, intermediate between NU and UC, is closer to the former than to latter. The mean value in recurrent PUNLMP is closer to UC than to NU. In NU, non-recurrent and recurrent PUNLMP, the acetylation to methylation ratio decreased from the nuclei in basal position to those in the surface, the average for the above groups being 1.491, 1.611 and 1.746, respectively. Setting the observed values for NU at each sampling location to unity, acetylation shows a steady decrease, the percentages of changes in this nuclear location compared to NU being −5% in non-recurrent PUNLMP, −15% in recurrent PUNLMP and −24% in UC. Concerning methylation, there is a slight increase in non-recurrent PUNLMP (+5%), a decrease in recurrent PUNLMP (-19%) followed by a sharp rise for the UC (+61%). In conclusion, there are differences in global histone acetylation and DNA methylation patterns between non-recurrent and recurrent PUNLMP. Further studies are needed to elucidate the complex interplay between chromatin structure, its modifications and recurrence of PUNLMP.