Flemming Brandt Sørensen

Growth and maturation of villi in placentae from well-controlled diabetic women

Summary Placentae from controls and two groups of diabetic women (one White classes A, B, C and the other classes D, F/R) were collected at 37–42 weeks of gestation. Tissue sections were analysed using stereological methods in order to quantify the growth and maturational status of villi. Birth and placental weights were recorded and placentae sampled in a systematic manner. Fields of view on formalin-fixed, paraffin-embedded sections were analysed to obtain estimates of volumes, surface areas, lengths and diffusion (harmonic mean) distances. Comparisons were drawn using three-way analyses of variance with group, mode of delivery and sex of newborn as the principal effects. Mean weights were similar in controls and diabetic groups. Diabetic placentae had a more voluminous fetal capillary bed of greater length, diameter and surface area. In addition, the diffusion distances across fetal plasma (erythrocyte to endothelium) were shorter. Stromal diffusion distance and villous diameter were greater in vaginal deliveries. Interaction effects influenced also villous capillarization, capillary volume, capillary diameter, trophoblast thickness and stromal thickness. Our results emphasize the importance of adaptations on the fetal side of the diabetic placenta. They show that changes can affect the placentae of appropriate-for-age as well as large-for-age babies and provide no evidence that they increase with the severity and duration of diabetes.

Stereological estimation of the mean and variance of nuclear volume from vertical sections

The application of assumption‐free, unbiased stereological techniques for estimation of the volume‐weighted mean nuclear volume, nuclear v̄V, from vertical sections of benign and malignant nuclear aggregates in melanocytic skin tumours is described. Combining sampling of nuclei with uniform probability in a physical disector and Cavalieris direct estimator of volume, the unbiased, number‐weighted mean nuclear volume, nuclear v̄N, of the same benign and malignant nuclear populations is also estimated. Having obtained estimates of nuclear volume in both the volume‐and number distribution of volume, a detailed investigation of nuclear size variability is possible. Benign and malignant nuclear populations show approximately the same relative variability with regard to nuclear volume, and the presented data are compatible with a simple size transformation from the smaller benign nuclei to the larger malignant nuclei. Finally, the variance in the volume distribution of nuclear volume is estimated by shape‐independent estimates of the volume‐weighted second moment of the nuclear volume, ***v̄2v, using both a manual and a computer‐assisted approach. The working procedure for the description of 3‐D size variability within benign and malignant nuclear populations can for all practical purposes be reduced to 2‐D measurement of nuclear profile areas. These new powerful stereological estimators of nuclear volume and nuclear size variability provide an attractive approach to quantitative and reproducible malignancy grading of cancers.

Systemic Administration of Insulin-Like Growth Factor I (IGF-I) Causes Growth of the Rat Prostate

PURPOSE To investigate the effects of insulin-like growth factor I (IGF-I) and epidermal growth factor (EGF) on the rat prostate. In addition, we investigated the effect of ornithine decarboxylase (ODC) inhibition with alpha-diflouromethylornitine (DFMO) on the expected growth of the prostate. MATERIALS AND METHODS Eight week old Wistar rats were allocated into groups of eight, receiving systemic treatment for 3 and 7 days with either IGF-I (400 micrograms/rat/day) or EGF (30 micrograms/rat/day) alone or in combination with DFMO. RESULTS Systemic treatment with IGF-I for 7 days resulted in a 29% (p < 0.01) increase in the mean wet weight of the ventral prostate. The mean weight of the dorsolateral lobe of the prostate increased by 39% (p < 0.05), and the seminal vesicle and coagulating gland increased 69% (p < 0.05) compared to controls. The ODC-activity in the prostate was significantly increased by IGF-I after 3 days of treatment, and administration of IGF-I concomitantly with DFMO significantly inhibited ODC activity and the weight increase of the prostate. Stereological examination of the prostate in the IGF-I-treated animals showed growth of the epithelial component of the gland. Systemic treatment with EGF did not affect the mean weight of the prostate or the seminal vesicle compared to controls. CONCLUSION The results demonstrate that treatment with IGF-I but not EGF for 7 days induces profound growth of the rat prostate and the seminal vesicle, and that the growth is dependent on an intact ODC-activity.

Objective histopathologic grading of cutaneous malignant melanomas by stereologic estimation of nuclear volume. Prediction of survival and disease‐free period

Modern stereologic techniques enable unbiased and shape-independent estimation of the three-dimensional nuclear volume (Vv). This study investigates the prognostic impact of Vv in 47 patients with malignant melanomas (10 years of follow-up) and compares Vv to traditional prognostic parameters and two-dimensional morphometric estimates. The averaged Vv was 226 microns3 and 457 microns3 in Stage I and II melanomas, respectively. The Vv was significantly increased in the case of ulceration, nodular melanoma, and Clarks level greater than III. The Vv showed only poor correlation to two-dimensional morphometric estimates. Cox regression analysis indicated Vv to possess excellent prognostic information, only rivaled by tumor ulceration, the latter being a 100% predictor of metastatic spread. Histologic type, Clarks level of invasion, tumor thickness (according to Breslow), and patient sex were without independent prognostic significance, which may be due to attributes of the small data base. It is concluded that Vv may be a powerful prognostic indicator in cutaneous melanomas, suitable for objective malignancy grading. The clinical and prognostic value of nuclear Vv needs further investigation in a larger and contemporary series of patients with malignant melanomas.

Oxygen diffusive conductance in placentae from control and diabetic women

SummaryRandom tissue sections of placentae from control and diabetic deliveries were analysed stereologically. The aim was to test whether or not adaptations in oxygen diffusive conductances occur to help compensate for fetal hypoxic stress in utero. Organs were from 34 control and 55 diabetic deliveries (39 from Whites classes A, B, C and 16 from classes D, F/R) arranged in two major groups (ABC and DFR). Tissue samples were fixed in formalin and processed for wax histology. Stereological and physicochemical quantities were used to calculate the partial conductances of six tissue layers, viz. maternal erythrocytes and plasma, villous trophoblast, villous stroma, fetal plasma and fetal erythrocytes. From partial conductances and birthweights, total and specific conductances for each placenta were determined. No differences were detected between diabetic placentae in different classes (A, B, C, D, F/R). However, both the ABC and DFR groups of diabetic placentae had higher total diffusive conductances than control organs. The increase was in the order of 7–25% and persisted even when adjustments were made for apparent differences in birthweights. The principal contributors to these changes were the post-trophoblastic (fetally located) tissue layers. These findings suggest that the diabetic placenta adapts to facilitate the diffusion of oxygen across the placenta and, thereby, to assist the hypoxic fetus.

DNA level and stereologic estimates of nuclear volume in squamous cell carcinomas of the uterine cervix. A comparative study with analysis of prognostic impact

Grading of malignancy in squamous cell carcinomas of the uterine cervix is based on qualitative, morphologic examination and suffers from poor reproducibility. Using modern stereology, unbiased estimates of the three‐dimensional, volume‐weighted mean nuclear volume (nuclear v̄Mv), were obtained in pretreatment biopsies from 51 patients treated for cervical cancer in clinical Stages I through III (mean age of 56 years, follow‐up period >5 years). In addition, conventional, two‐dimensional morphometric estimates of nuclear and mitotic features were obtained. DNA indices (DI) were estimated by flow cytometry. Finally, the semiquantitative malignancy grade score value (MGS) was determined according to previously published methods. Estimates of nuclear v̄Mv were on average increased in euploid lesions (2P = 0.01), but the overall relationship between nuclear v̄Mv and DI was poor. Different clinical stages of disease did not differ with regard to nuclear v̄Mv (2P = 0.99) and DI (2P = 0.56). No relationship was disclosed between MGS and nuclear v̄Mv (2P = 0.85). Single‐factor analysis showed prognostic impact of clinical stage of disease (2P = 0.0001) and DI (2P = 0.04), whereas estimates of nuclear v̄Mv were only of marginal prognostic significance (2P = 0.07). How‐ M ever, Cox multivariate regression analysis showed independent prognostic value of patient age and nuclear v̄Mv along with clinical stage and DI. All other investigated variables were rejected from the model. A prognostic index with highly distinguishing capacity between prognostically poor and favorable cases was constructed (2P = 1.9 × 10−7). It is concluded that realistic estimates of nuclear volume are independent of nuclear DNA content and are of prognostic value for objective malignancy grading in patients with squamous cell carcinoma of the uterine cervix.

A note on stereological estimation of the volume-weighted second moment of particle volume

It is shown that for a variety of biological particle shapes, the volume‐weighted second moment of particle volume can be estimated stereologically using only the areas of particle transects, which can be estimated manually by point‐counting.

Quantitative histopathology in the prognostic evaluation of patients with transitional cell carcinoma of the urinary bladder

Background. Morphologic grading of malignancy is considered to be of prognostic value in patients with transitional cell carcinomas of the urinary bladder (TCC). This qualitative approach is, however, associated with low reproducibility. Grading of malignancy can be carried out on a reproducible, quantitative scale.

Quantitative histopathological variables in in situ and invasive ductal and lobular carcinomas of the breast

This study was carried out to compare quantitative histopathological estimates obtained in normal breast epithelium (N = 15), lobular carcinoma in situ (N = 29), ductal carcinoma in situ (N = 24), invasive lobular carcinoma (N = 39), and invasive ductal carcinoma (N = 71) of the female breast. Using unbiased stereology, the three‐dimensional mean nuclear size, v̄v (nuc), was estimated in routine histological sections, along with morphometric point‐counting based estimates of the mean nuclear profile area, āH (nuc). and estimates of the nuclear density index, NI, the mitotic index, MI, and the nuclear volume fraction, Vv (nuc/tis). The v̄v (nuc), āH (nuc), and MI were, on average, larger in ductal than in lobular carcinomas (2p≤0.01), whereas the mean NI was smaller in ductal carcinomas (2p= 3 · 10‐4). Comparing estimates obtained in tumors of pure ductal carcinoma in situ (N = 11) with those obtained in tumors of pure lobular carcinoma in situ (N = 7), only the difference in mean NI reached statistical significance (2p= 0.001). Several significant differences were found between means of quantitative histopathological estimates obtained in normal breast epithelium, pure in situ lesions, and invasive carcinomas. Overlaps were, however, evident among the groups. There were no significant differences between means of the quantitative variables obtained in carcinoma in situ of the ductal and the lobular type with or without accompanying invasive carcinoma (2p≥0.22). A close correlation was found between estimates of v̄v (nuc) obtained in the in situ component and the invasive part of ductal carcinomas (r= 0.86, 2p= 2 · 10‐4). Previous studies have shown prognostic value of quantitative histopathological variables in breast carcinomas. The present study points to an additional value of the investigated variables in the diagnostic separation of normal breast epithelium, in situ lesions, and invasive carcinomas. The quantitative variables obtained in the in situ lesions did not indicate whether an accompanying invasive tumor was present or not.

Stereological estimates of nuclear volume in squamous cell carcinoma of the uterine cervix and its precursors

Using modern stereology, this study was carried out to obtain base-line data concerning three-dimensional, mean nuclear size in precancerous and invasive lesions of the uterine cervix. Unbiased estimates of the volume-weighted mean nuclear volume (nuclear ¯v v were obtained by point-sampling of nuclear intercepts in 51 pre-treatment biopsies from patients with invasive squamous cell carcinomas (SCC). Vertical sections from 27 specimens with cervical intraepithelial neoplasia (CIN) grades I through III were also investigated, along with 10 CIN III associated with microinvasion (CIN III+M). On average, nuclear ¯v v was larger in SCC than in CIN III and CIN III + M together (2P=8.9·10−5). A conspicuous overlap of nuclear ¯v v existed between all investigated lesional groups. The reproducibility of estimates of nuclear ¯v v in biopsies with SCC was acceptable (r=0.85 andr=0.84 in intra- and inter-observer studies, respectively). The efficiency of the sampling scheme was high, with more than 60% and more than 80% of the total observed variance contributed by differences between individual lesions with CIN and SCC, respectively. Estimates of nuclear ¯v v based on sampling within the whole epithelial thickness and on sampling in the lower one-third in CIN I and the lower two-thirds in CIN II lesions were of the same magnitude. Approximate estimates of the absolute variation of nuclear ¯v v were directly proportional to individual estimates of nuclear ¯v v, whereas the relative variation of nuclear ¯v v tended to decrease with increasing mean nuclear volume. Based on the rather small number of cases investigated, estimates of nuclear ¯v v are unable to distinguish between different grades of CIN. However, the estimation of nuclear ¯v v is well-suited for the purposes of objective grading of malignancy in SCC.