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PubMed1991

Paragangliomas: neuroendocrine features and cytometric DNA distribution patterns. A clinico-pathological study of 22 cases.

Paragangliomas from 22 patients with extraadrenal tumours of this type were studied. Neuroendocrine features were examined using immunohistochemical techniques. Twenty-two antisera raised against neuroendocrine "markers", regulatory peptides, serotonin and intermediate filament proteins were studied in this group and cytometric DNA assessments were made by means of image cytometry. One normal and 5 hyperplastic carotid bodies were used as controls in the DNA cytometric investigations. Clinical and/or histopathological evidence of "malignancy" was present in 5 cases. The tumour cells showed heterogeneity with regard to their expression of different peptides, and the immunohistochemical analyses did not permit differentiation between benign and malignant paragangliomas. An euploid nuclear DNA distribution pattern was found in all controls and in 17 of the tumours; all except 1 were clinico-pathologically benign. An aneuploid DNA pattern was observed in 5 of the cases and some malignant features were present in 4 of these cases. DNA data may give further information apart from that obtained from the histopathological findings which may be of value in predicting the biological behaviour of this tumour type.

Adult↗
PubMed1991

Nuclear DNA content in 27 pancreatic endocrine tumours: correlation with malignancy, survival and expression of glycoprotein hormone alpha chain.

Paraffin-embedded tissue from resection specimens of 14 functioning and 13 nonfunctioning pancreatic endocrine tumours (PET) was analysed for nuclear DNA content by image cytometry. Data on follow-up (mean 5.5 years) were available in all patients. DNA histograms with a diploid pattern were found in 13 (48%) tumours, while an aneuploid pattern was seen in the remaining 14 tumours (52%). Six (40%) of the diploid tumours and 9 (60%) of the aneuploid tumours were malignant. Survival was shorter in patients with malignant and aneuploid PET (mean 3.5 years, range 0.5-7) than in those with malignant and diploid PET (mean 5.7 years, range 3-8). Human chorionic gonadotropin-alpha was expressed in 3 of 12 benign PET, with 1 being aneuploid, and 6 of 15 malignant PET, with 4 being aneuploid. We conclude from these results that the ploidy pattern of PET allows no discrimination between benign and malignant tumours but may provide prognostic information on the aggressiveness of malignant PET.

Adult↗
PubMed1996

Cell cycle expression of estrogen receptors determined by image analysis on human breast cancer cells in vitro and in vivo.

We have investigated, by image analysis, the cell cycle expression of estrogen receptors (ER) on MCF-7 cell line and on MCF-7 xenografts. The results demonstrate, in vitro as well as in vivo, an increase of ER concentration during the G0/G1-phase, followed by a decrease during the S-phase until the late S-phase where a rapid increase was noted. These results confirm that estrogens are involved in the DNA synthesis since ER is expressed in vivo at a maximal level in the late G1. In presence of saturating concentrations of 17 beta-estradiol, the mean ER concentration in G0/G1 phase is significantly decreased compared with the control cells cultured in estrogen-deprived medium. This indicates that 17 beta-estradiol down-regulates ER preferentially in the G0/G1 phase. These data suggest that ER in S and G2/M phases is unable to interact with its ligand. Consequently, estrogens may have no effects on the entry of cells in mitosis. Finally, after long-term tamoxifen treatment of MCF-7 xenografts, a tamoxifen-resistant tumor was developed which was characterized by a change in the profile of ER concentration during the G0/G1 phase. In conclusion, it is possible that the differences in cell cycle distribution of ER could be correlated with different phenotypes of breast cancer and also with different clinical phases of tumoral evolution. However, it remains to be known what is the clinical significance of the ER cell cycle expression in relation to tumor aggressiveness and survival.

Animals↗
PubMed1995

Chemotherapy-induced nuclear alterations of morphologic and genomic characteristics in a human colon cancer grafted onto nude mice.

PURPOSE: A human Dukes B colonic adenocarcinoma was grafted onto 40 nude mice. The mice were divided into four groups, one control and three representing experimental conditions. Animals in the three experimental groups received either adriamycin (ADR), 5-fluorouracil (5-FU), or camptothecin (CPT) over a 25-day period beginning 34 days after grafting. Control animals received saline on an identical schedule. Animals were killed 105 days after grafting. METHODS: The effect of therapy was assessed by three techniques: 1) tumor size was periodically measured during the life of the animals, 2) modifications of APC, Ki-ras, and p53 genes were studied by polymerase chain reaction, dot-blot analysis, restriction analysis, and DNA sequencing, and 3) image cytometry of Feulgen-stained material was used to characterize 15 parameters describing morphometric, densitometric, and textural features of tumor nuclei. RESULTS: When compared with controls, tumor growth (size) was maximally suppressed by treatment with CPT (P < or = 0.001). Growth was inhibited significantly by treatment with 5-FU (P < or = 0.01); no statistical difference in tumor size was observed between controls and animals treated with ADR. Modifications of APC, Ki-ras, and p53 genes were not observed; however, treatment did inhibit amplification of APC and p53 genes. CONCLUSIONS: The 15 morphonuclear parameters were assessed to define populations of cell nuclei altered by chemotherapy. Although CPT maximally suppressed growth, it did not alter nuclear morphology when compared with controls. Treatment with either 5-FU or ADR resulted in nuclear morphologic alterations defined as distinct populations using multivariate analysis. Nonsupervised linear discriminant analysis was used to quantify the relative proportions of these populations. Four morphonuclear parameters were identified, which discriminated nuclei exposed to either ADR or 5-FU from controls.

Adenocarcinoma↗
PubMed1991

Multiple primary colorectal adenocarcinomas: cytometric DNA ploidy patterns and histopathologic features.

The nuclear DNA distribution pattern was studied by means of image cytometry in 18 patients with synchronous and 7 patients with metachronous colorectal adenocarcinomas. About 80 percent of a total number of 53 carcinomas were found to consist of neoplastic cells where the nuclear DNA distribution pattern was of the aneuploid type. In 19 of the 25 patients, all carcinomas in the individual colon had identical nuclear DNA distribution patterns. Aneuploid tumors tended to appear more frequently in the sigmoid colon and the rectum than in the right colon. Carcinomas associated with an adenoma were more common in the rectum and in Dukes' A carcinomas. The results of cytometric assessments of the nuclear DNA ploidy pattern of the neoplastic cells in colorectal carcinomas have previously been found to be an independent prognostic variable. From the observations made in the present study, it is concluded that the cytometric DNA ploidy pattern of multiple primary colorectal carcinomas corresponds to that found in single carcinomas. The similarity between the two groups of cancer patients is also supported by conformity between histopathologic features and prognosis.

Adenocarcinoma↗
PubMedDate not supplied

Prognostic factors in parathyroid cancer: a review of 95 cases.

The clinical course, histopathology, and tumor DNA distribution patterns were analyzed in 95 patients with parathyroid cancer. The median follow-up was 6 years (range 1-25 years). Eighteen patients received a benign diagnosis at their first operation. The initial procedure was tumor resection in 42 patients and tumor resection plus partial or total thyroidectomy in 40 patients. Forty patients developed recurrent disease and 36 patients underwent 1 to 9 re-operations. Cervical recurrence and lung metastases were most commonly encountered. The median time from the first operation to recurrence was 33 months (range 1-228 month). Twenty-one patients died of parathyroid cancer a median of 28 months following discovery of their first recurrence. The histopathological reevaluation confirmed unequivocal parathyroid cancer, i.e., infiltration and/or metastases, in 41 cases. Fifty-four cases lacked these criteria but showed various forms of atypia. Image cytometry demonstrated tumor aneuploidy in 26 of 39 cases with definite cancer by histological criteria, compared to the 13 of the 52 with equivocal histological diagnosis. Twelve patients with aneuploid tumors and 7 patients with euploid tumors died of parathyroid cancer. In a multivariate analysis, patients treated with extensive surgery, i.e., tumor resection and unilateral or bilateral thyroidectomy, had a longer survival and a longer relapse-free period. Other factors of importance for survival were age and histopathology. Histopathology and an aberrant nuclear DNA content were important factors for the time to recurrence. We conclude that histopathology alone is unable to confirm a cancer diagnosis in the absence of infiltration and/or metastases. Because recurrence may occur late, patients should be followed closely. Even repeated surgical interventions have proven beneficial.

Actuarial Analysis↗
PubMedDate not supplied

Segmentation of sperms using the strategic Hough transform.

A new method based on the Hough transform has been used for the quantitative estimation of the morphological characteristics of the sperm. Images of the sperms were acquired into the digital format using the optical microscope, CCD camera, and frame grabber. For each sperm in the image, the region of interest for the segmentation of the sperm head was selected using the density difference between the sperm head and background. The boundary of the sperm head was approximated with an ellipse and represented by five parameters. These five parameters representing an ellipse have been investigated by applying the Hough transform strategically. During the searching procedure using the Hough transform, we restricted the searching volume minimally and moved the next searching volume to the direction showing the steepest gradient of the estimation error. Also, we increased the parameter resolution from the three pixel distance to the one pixel distance. For the tested images, head boundaries of the sperms were estimated with an error of 1/2 pixel distance, and morphological parameters were calculated by the equations without further image processing. This new method separates sperm head based on the geometrical characteristic of the sperm and calculates morphological parameters simply from the derived boundary.

Algorithms↗
PubMed1995

Combined use of nuclear morphometry and DNA ploidy as prognostic indicators in nonresectable adenocarcinoma of the pancreas.

The introduction of combined modality treatment has only marginally affected the prognosis in nonresectable pancreatic carcinoma. Evaluation of easily accessible prognostic variables could be of clinical importance when selecting patients for proper therapy. DNA ploidy and morphometric variables were chosen as prognostic markers and assessed on cytologic material obtained by fine-needle aspiration biopsy (FNAB) from 128 patients with pancreatic carcinoma. The nuclear DNA content was measured by image cytometry. Patients were categorized as short-term (< or = 6 mo) and long-term survivors (> 6 mo). Ninety-eight of 116 nonresectable patients were possible to evaluate. There were significant differences between short- and long-term survivors with regard to DNA ploidy (p < 0.01) and the morphonuclear variable anisokaryosis (p < 0.001). In patients with either DNA aneuploid tumors or anisokaryosis > or = 0.6, the survival time was 6 mo or less in 85 and 93%, respectively. When both these criteria were fulfilled, only 5% survived for more than 6 mo. Thus, DNA ploidy and morphometry, separately or in combination, may provide prognostic information in nonresectable pancreatic carcinoma.

Adenocarcinoma↗
PubMed1995

DNA Ploidy Pattern in Pure Seminomas.

Nuclear extract and image cytometry was used to determine the DNA ploidy pattern of 31 pure seminomas. At least 5-year but usually 10-year follow-up was available to compare the clinical outcome to the DNA ploidy pattern. In 24 cases (77.4%) the DNA indexes (DI) showed tetraploid (1,8 < DI < 2,2) pattern confirming the recent cytogenetic and flow cytometric DNA studies of others. However, in 7 cases (22,6%) the tumors were aneuploid. Out of these aneuploid cases 2 had two subpopulations and 2 had less than 1,8 DI. These latter cases (providing the loss of chromosomal DNA) had elevated AFP levels in serum that raises the question of nonseminomatous transformation without any morphological evidence. Usually, the aneuploid cases had worse prognoses but there was no significant difference. Because of the small number of aneuploid cases wider clinicopathologic studies are required to confirm our results.

Journal Article↗
PubMed1998

Measurement of apoptosis.

The cell dying by apoptosis undergoes a sequence of morphological, biochemical, and molecular changes which are characteristic, and often unique, to this mode of cell death. Specific features of apoptotic cells resulting from these changes, which serve as markers used to reveal the apoptotic mode of cell death and to quantify the extent of apoptosis in cultures or in tissue, are reviewed. Analysis of these features by flow or image cytometry is the most commonly used approach to detect, quantify, and study various aspects of apoptosis. Flow or laser scanning cytometry also offer all the advantages of rapid, accurate and multiparametric measurements to investigate the biological processes associated with cell death. Numerous methods have been developed to identify apoptotic and necrotic cells, which are widely used in various disciplines, particularly in oncology and immunology. The methods based on changes in cell morphology, plasma membrane molecular structure and transport function, function of cell organelles, DNA stability to denaturation and endonucleolytic DNA degradation are reviewed and their applicability in the research laboratory and in the clinical setting is discussed. The most common pitfalls and improper use of the methodology in analysis of cell death and in data interpretation are also discussed.

Animals↗
PubMed1996

[Comparative DNA cytometric and cytogenetic ploidy determination of chondrosarcoma and osteosarcoma].

10 chondrosarcomas and 10 osteosarcomas were examined using cytogenetics and DNA-image-cytometry. Cytogenetically 6 of 10 chondrosarcomas and 4 of 10 osteosarcomas showed hyperdiploid tumorcells. By DNA-cytometry in 8 of 10 chondrosarcomas and 9 of 10 osteosarcomas hyperdiploid tumorcells resp. hyperdiploid stemlines were detected. This discrepancy reflects an in-vitro-selection depending on the different entities. In 7 aneuploid clones of chondrosarcomas the chromosomal ploidy was calculated using the relative length of the chromosomes and compared with the DNA-ploidy of the native tumor. There was a close relation between both parameters of nuclear DNA-content. The interpretation of cytogenetic results is improved using a combination of karyotypic and DNA-cytometric examination. This is particularly important for the search for relations between numeric chromosomal aberrations and morphological parameters (grading).

Aneuploidy↗
PubMed1996

[Carcinoma-in-carcinoma metastasis. In situ DNA quantification and immunohistochemical diagnosis].

In the rare event of cancer-to-cancer metastasis, one malignant neoplasm spreads to a second carcinoma. To date, 42 such cases have been published in the English- and German-language literature. We report two additional patients, each with metastatic bronchial cancer present in a renal carcinoma. In addition to immunohistochemical examinations, we performed in situ DNA image cytometry of histologic preparations as a novel analytical technique to differentiate metastatic tissue from the host tumor. In both cases DNA histograms of metastatic tumor resembled those of the primary malignancy but were clearly dissimilar to the host tumor. In 70% of previously reported cases renal carcinoma was the host tumor while in 50%, cancer of the lung was the most frequent donor tumor.

Adenocarcinoma↗
PubMed1997

[Serous psammocarcinoma of the ovary. Case report and literature review].

We report a rare case of psammocarcinoma which presented as a mass in the lower abdomen of an otherwise asymptomatic woman. Ultrasonographically, the tumor showed extensive calcification and was suspected to be a myoma. DNA image cytometry and assessment of proliferative activity were performed and showed an euploid, low proliferating tumor cell population. This underlines the low malignancy of this type of carcinoma, which is comparable to that of serous borderline lesions of the ovary and explains the benign clinical course.

Calcinosis↗
PubMed1999

The Feulgen reaction 75 years on.

The Feulgen reaction proposed by Feulgen and Rossenbeck 75 years ago is one of the cytohistochemical reactions most widely used in biology and medicine. It allows DNA in situ to be specifically stained based on the reaction of Schiff or Schiff-like reagents with aldehyde groups engendered in the deoxyribose molecules by HCl hydrolysis. The staining intensity is proportional to the DNA concentration. Current applications of the Feulgen reaction are mainly concerned with DNA quantification in cell nuclei by image cytometry for ploidy evaluation in tumor pathology. From the morphological point of view, specific demonstration of DNA in cell structures at the light microscopic level is very little used nowadays. On the other hand, application of the Feulgen principles to electron microscopy have recently allowed specific DNA-staining procedures to be developed for the study of the structural organization of DNA in situ.

Animals↗
PubMed2006

Morphometric sum optical density as a surrogate marker for ploidy status in prostate cancer: an analysis in 180 biopsies using logistic regression and binary recursive partitioning.

We sought to identify morphometric descriptors predictive of nondiploidy in prostatic adenocarcinoma on prostate needle biopsies using logistic regression (LR) and binary recursive partitioning (BRP) and compare the equivalence of both methods. A total of 180 prostate needle biopsies diagnosed as prostatic adenocarcinoma were selected. Deoxyribonucleic acid ploidy and morphometry were performed separately on Feulgen-stained sections from these biopsies using the CAS-200 system and Nuclear Morphometry Suite, respectively. Seven morphometric predictors were tested as predictor variables, including nuclear area, circularity, elongation, sum optical density (OD), configuration run length, coefficient of variation (CV), and angularity. Logistic regression (LR) identified a two-parameter model including sum OD and circularity that had a 93.9% overall correct prediction rate (area under curve=0.950; 95% CI: 0.913, 0.987). A reduced model including only sum OD was equally good without any significant loss of predictive accuracy (93.3% correct overall classification rate). BRP also selected sum OD as the most predictive parameter; a sum OD cut-off of 7.73 in this model identified 93.3% of the nondiploid cases correctly. Morphometric OD can be used as a surrogate marker of nondiploidy. LR and BRP models are both equivalent in identifying and correctly classifying nondiploid cases of prostate cancer using sum OD as the predictor variable.

Adenocarcinoma↗
PubMed2001

Improvement of breast cancer prognostication using cell kinetic-based silver-stainable nucleolar organizer region quantification of the MIB-1 positive tumor cell compartment.

Recently, it was stated that the proliferative activity (P) of a cell population could be indirectly calculated by multiplying the MIB-1 immunopositivity and silver-stainable nucleolar organizer region (AgNOR) features extracted exclusively in MIB-1 positive (pos.) nuclei: P=MIB-1 x AgNOR(MIB-1pos.). To study the prognostic significance of this hypothesis, MIB- immunohistochemistry and AgNOR staining were applied on a series of 89 cases of breast cancer with an 8-year follow-up period. The mean MIB-1 immunopositivity (MIB-1mean) was evaluated immunohistometrically on paraffin sections using a TV image analysis system CM-2 (Hund, Wetzlar, Germany). Later, a combined MIB-1/AgNOR staining was applied and evaluated using a TV image analysis system AMBA (IBSB, Berlin, Germany). The AgNOR features of 150 randomly chosen tumor nuclei were investigated, irrespective of their MIB-1 status (AgNOR count, AgNOR area). Later, a second measurement was performed on 100 MIB-1 positive tumor nuclei exclusively (AgNOR countMIB-1pos., AgNOR areaMIB-1pos.). AgNOR count and AgNOR countMIB-1pos. showed a different data distribution [2.7+/-0.7 (mean+/-SD) vs 3.9+/-1.1; r=0.315, P=0.014]. Similar results were obtained for AgNOR area and AgNOR areaMIB-1pos. (5.1+/-2.1 microm2 vs 7.5+/-2.4 microm2; r=0.501, P<0.001). Kaplan-Meier survival curves revealed significant differences for MIB-1mean (P=0.0018) and AgNOR areaMIB-1pos. (P=0.0340). In Cox models, both parameters provided independent prognostic information. Using their combination, the P, three groups of patients with statistically different survival could be separated (P=0.0014). Thus, the combination of MIB-1-immunopositivity and AgNOR measurements in MIB-1 positive nuclei appears to be more useful in breast cancer prognosis than the exclusive application of one of the two methods. By this combined application, probably effects of tumor biology are represented more precisely.

Adenocarcinoma↗
PubMed2006

Voxel-based morphometry indicates relative preservation of the limbic prefrontal cortex in early Huntington disease.

In Huntington disease (HD), both the genetic defect and mutant gene product huntington are known but the exact mechanisms that lead to neuronal loss are poorly understood. Until now, the distribution of tissue loss throughout the brain has been investigated intensively. Here we searched for areas that, antipodal to the striatum, display grey-matter (GM) preservation. We performed high resolution T1-weighted magnetic resonance imaging and voxel-based morphometry in 46 patients in early HD and 46 healthy controls. We applied an analysis of covariance (ANCOVA) model with the total GM volume of each participant as covariate. In accordance with earlier reports, group comparisons revealed GM decrease in the striatum, insula, and thalamus as well as in dorsolateral frontal and occipital areas. In contrast, the limbic prefrontal cortex displayed GM preservation. Our findings support hypotheses that postulate differential involvement of frontosubcortical circuits in the pathophysiology of HD.

Adult↗
PubMed2005

A computer reconstruction of the entire coronary arterial tree based on detailed morphometric data.

A rigorous analysis of blood flow must be based on the branching pattern and vascular geometry of the full vascular circuit of interest. It is experimentally difficult to reconstruct the entire vascular circuit of any organ because of the enormity of the vessels. The objective of the present study was to develop a novel method for the reconstruction of the full coronary vascular tree from partial measurements. Our method includes the use of data on those parts of the tree that are measured to extrapolate the data on those parts that are missing. Specifically, a two-step approach was employed in the reconstruction of the entire coronary arterial tree down to the capillary level. Vessels > 40 microm were reconstructed from cast data while vessels < 40 microm were reconstructed from histological data. The cast data were reconstructed one-bifurcation at a time while histological data were reconstructed one-sub-tree at a time by "cutting" and "pasting" of data from measured to missing vessels. The reconstruction algorithm yielded a full arterial tree down to the first capillary bifurcation with 1.9, 2.04 and 1.15 million vessel segments for the right coronary artery (RCA), left anterior descending (LAD) and left circumflex (LCx) trees, respectively. The node-to-node connectivity along with the diameter and length of every vessel segment was determined. Once the full tree was reconstructed, we automated the assignment of order numbers, according to the diameter-defined Strahler system, to every vessel segment in the tree. Consequently, the diameters, lengths, number of vessels, segments-per-element ratio, connectivity and longitudinal matrices were determined for every order number. The present model establishes a morphological foundation for future analysis of blood flow in the coronary circulation.

Algorithms↗