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PubMed1995

Malignancy-associated changes in the breast. Changes in chromatin distribution in epithelial cells in normal-appearing tissue adjacent to carcinoma.

Malignancy-associated changes (MACs) have been described as subtle morphologic changes in normal-appearing tissues adjacent to or distant from malignant tumors. MACs might be applicable as a marker of occult malignancy, increased risk of subsequent carcinoma or recurrence of tumor after therapy. The aim of this study was to verify the existence of MACs in normal-appearing breast tissue adjacent to breast carcinoma. Image cytometry measurements were performed on 4-microns sections cut from formaldehyde-fixed tissue blocks and stained with thionin-SO2 following the Feulgen procedure. Morphologically normal appearing breast lobules were analyzed in patients with benign (20 cases) and malignant breast disease (23 invasive and 11 in situ tumors). The chromatin distribution features of the epithelial nuclei in normal-appearing breast lobules differed between patients with benign and malignant breast disease. Changes in nuclear features, detected in epithelial nuclei from normal-appearing lobules in breasts resected for carcinoma, were defined as MACs. The frequency of MAC nuclei was low in benign tissues, increased in tissues with ductal carcinoma in situ and highest in tissues with invasive carcinoma. Based on the measurements of nuclei in normal-appearing lobules, it was possible to discriminate between patients with benign breast disease and patients with invasive carcinoma in 86% of cases.

Breast↗
PubMed1998

DNA ploidy determination of early molar pregnancies by image analysis: comparison to histologic classification.

BACKGROUND: To improve histologic diagnosis of molar pregnancies, updated guidelines have been proposed recently. These guidelines take into account that less developed molar gestations differ from their fully developed counterparts. OBJECTIVE: To test the validity of these criteria by correlating histologic diagnosis with ploidy determination accomplished by means of image analysis. DESIGN: Fifty archival cases of early molar pregnancy were reclassified according to the new criteria. The diagnosis had to be changed from partial to complete hydatidiform mole (PM to CM, respectively) in 9 cases and from CM to PM in 4 cases. DNA image cytometry could be performed in 40 cases (CM, n = 21; PM, n = 19). RESULTS: There was 100% agreement between histologic diagnosis and a diploid or polyploid histogram in CM and 79% agreement between triploidy and PM, when the updated diagnostic criteria were used. This represents an improvement compared with diagnoses made with former criteria. Nevertheless, problems of correct classification remain: In 3 cases classified as PMs, fetal remnants were accompanied by the histologic appearance of a CM. These 3 cases could represent either a true embryonic development in CM or a twin gestation with one normal pregnancy and one mole, or they could belong to a (very rare) third type of mole. All of them show the same risk of persistent trophoblastic disease observed in classic CM. CONCLUSIONS: As the groups at risk for developing persistent trophoblastic disease can be identified by their DNA histograms, ploidy analysis would be desirable in addition to histologic examination.

Adult↗
PubMed1994

Malignancy associated changes in squamous epithelium of the head and neck region.

Subtle cellular changes are known to exist in normal host tissue adjacent to tumours. These are called malignancy associated changes (MAC). To get more insight into the degree of expression and local spread of such changes we performed high resolution image cytometry on visually normal intermediate cell nuclei in smears from patients with laryngeal or pharyngeal squamous cancer. The smears were taken from the tumour surface, from a border region of the tumour and from a distant unsuspicious buccal site. In addition buccal smears from healthy control persons were examined. In a pilot study smears from 12 cancer patients and 11 control persons and in a succeeding validation study 63 controls, 18 non-tumour patients and 25 cancer patients were investigated. In both studies the occurrence of MACs was demonstrated quantitatively. In cancer patients normal appearing intermediate cells from the three different sampling sites could be discriminated with 65% in the pilot study and with 53% correct classification in the validation study. In addition the influences of smoking behaviour and sex were investigated in the control group. Only in the latter case there was a significant difference between female and male with a 63% correct cell and 71% correct specimen classification.

Carcinoma, Squamous Cell↗
PubMed1990

Variations in Feulgen stainability of epithelial parenchymal cells extracted from paraffin-embedded salivary gland specimens.

The variations of Feulgen stainability of cells extracted from paraffin-embedded archival specimens for DNA assessment by means of image cytometry (ICM) were investigated in normal salivary gland parenchyma. The Feulgen stainability of the deparaffinized, rehydrated, and disaggregated preparations was found to exhibit variations of up to 300%, expressed by the mean of integrated optical density (IOD), when a routine procedure was applied to a first series of Cytospin preparations of disaggregated specimens. When measured in nondisaggregated tissue sections, only negligible variations were observed. After minimization of the mechanical strains to the cellular material in the Cytospin preparations in a second series, the variations in Feulgen stainability were found to be considerably lower. The findings indicate that the main reason for variations in the Feulgen stainability of extracted cells is, most likely, the disaggregation procedure itself. Factors such as initial treatment of the specimens, duration and kind of formalin fixation, and length of storage time periods seem to be of minor importance. Retrospective studies on paraffin-embedded specimens require a carefully controlled tissue type-adapted disaggregation procedure. In addition, we concluded that the interpretation of histograms, obtained by means of ICM DNA assessments in Cytospin preparations of archival material, requires a well-defined internal specific standard.

Coloring Agents↗
PubMed1993

Characteristics of DNA replication in isolated nuclei initiated by an aprotinin-binding protein.

Isolated cell nuclei were used as the source of template DNA to investigate the role of a cytosolic aprotinin-binding protein (ADR) in the initiation of eukaryotic DNA replication. Computerized image cytometry demonstrated that the DNA content of individual nuclei increased significantly following incubation with ADR-containing preparations, and the extent of DNA synthesis is consistent with that allowed by the limiting concentration of dTTP. Thus, dTTP incorporation into isolated nuclei represents DNA synthesis and not parent strand repair. We found that dTTP incorporation into the isolated nuclei is dependent on DNA polymerase alpha (a principal polymerase in DNA replication) but that DNA polymerase beta (a principal polymerase in DNA repair processes) does not play a significant role in this system. Finally, neither aprotinin nor a previously described cytosolic ADR inhibitor can block the replication of nuclease-treated calf thymus DNA, while both strongly inhibit replication of DNA in isolated nuclei. This result, coupled with the relative ineffectiveness of nuclease-treated DNA compared with nuclear DNA to serve as a replicative template in this assay, argues against a significant contribution from repair or synthesis which initiates at a site of DNA damage. These data indicate that ADR-mediated incorporation of 3H-dTTP into isolated nuclei results from DNA replicative processes that are directly relevant to in vivo S phase events.

Aprotinin↗
PubMed1991

Ki-67 labeling in postmitotic cells defines different Ki-67 pathways within the 2c compartment.

Simultaneous quantification of DNA and Ki-67 proliferation-associated antigen was performed using fluorescence image cytometry. In the MCF-7 cell line, the Ki-67 antigen content increases during the cell cycle, and its intranuclear distribution pattern varies. Quantitative evolution of Ki-67 content as a function of nuclear area makes it possible to define several pathways followed by cells going through the 2c compartment. 1) In some cells, the amount of Ki-67 antigen remains constant during G1 (Ki-67 stable pathway), and a characteristic speckled pattern can be observed. 2) In the larger fraction of cells analyzed, there is a postmitotic decrease in the Ki-67 (Ki-67 decrease pathway) content. In this pathway, labeling is located in the nucleoplasm in small nuclei, is located in nucleoli in intermediate-sized nuclei, and is absent from larger nuclei (G0). A progressive increase in Ki-67 content (Ki-67 increase pathway) was observed from intermediate-sized nuclei to S phase nuclei. From these results, we hypothesize that the Ki-67 stable pathway is the G1 phase of newly formed cells going directly to S phase in local optimal conditions of growth and that Ki-67 decrease pathway and Ki-67 increase pathway correspond to cells whose progression to S phase is regulated by extracellular factors.

Animals↗
PubMed2005

Characterisation of breast fine-needle aspiration biopsies by centrosome aberrations and genomic instability.

Recent studies have suggested that aneuploidy in malignant tumours could be a consequence of centrosome aberrations. Using immunofluorescence analysis with an antibody against gamma-tubulin and DNA image cytometry, we measured centrosome aberrations and DNA ploidy patterns in fine-needle aspiration biopsies (FNABs) of 58 breast lesions. Benign lesions did not show any centrosome aberrations. DNA diploid carcinomas showed a mean percentage of cells with centrosomal defects of 2.1%. The aneuploid invasive carcinomas could be divided into two subgroups by their significantly (P=0.0003) different percentage of cells with centrosome aberrations (2.0 and 10.3%, respectively) and their significantly (P=0.0003) different percentage of cells with nonmodal DNA content values determined by the Stemline Scatter Index (SSI), a measure of genomic instability. The percentage of cells with centrosome aberrations demonstrated a positive, linear correlation with the corresponding SSI (r=0.82, P<0.0001) and loss of tissue differentiation (r=0.78, P<0.0001). Our results indicate the percentage of cells with centrosome aberrations as being sufficient to divide the investigated tumours into three significantly different groups: benign lesions with no centrosomal aberrations, and two malignant tumour types with mean values of 2.1 and 9.6% of centrosomal defects, respectively. Together, these results demonstrate that centrosome aberrations correlate with genomic instability and loss of tissue differentiation. Furthermore, this study shows the feasibility of centrosomal analysis in FNAB of the breast and suggests centrosomal aberrations as possessing diagnostic and prognostic value.

Adult↗
PubMed2005

Cell cycle and/or proliferation markers: what is the best method to discriminate cervical high-grade lesions?

The aim of this study on a series of biopsies diagnosed as normal, metaplastic, low-grade squamous intraepithelial lesions (LSILs), and high-grade squamous intraepithelial lesions (HSILs) was dual: to determine the chronology of cell cycle and proliferation abnormalities after human papillomavirus infection during the development of squamous intraepithelial lesions and to determine the best diagnostic indicator(s) linked to the appearance of an HSIL. Ninety-nine cervical biopsies, 18 normal, 9 with metaplastic changes, 29 LSIL, and 43 HSIL (23 cervical intraepithelial neoplasia 2 and 20 cervical intraepithelial neoplasia 3), were analyzed by image cytometry for DNA ploidy and p16INK4A determination, AgNOR counting, MIB-1, and ICBP90 immunostaining quantification. The human papillomavirus status had been previously determined on corresponding cytological smears with the Hybrid Capture II test. Suspect DNA profile and p16INK4A staining were the first significant events that preceded the increase of cell proliferation. Indeed, these markers were the best tests for the detection of a lesion, whatever its grade (positive predictive values of 90% and 100%, respectively). The presence of MIB-1- or ICBP90-positive cells in the upper two thirds of the epithelium was a very accurate feature to select HSIL (sensitivity, 100% for MIB-1) but with a low specificity. The sensitivity of a suspect DNA profile associated with a positive MIB-1 or ICPB90 immunostaining for the detection of an HSIL was, respectively, 92.8% and 92.7%; their specificities were 54.2% and 44%; their positive predictive values were 78% and 73%; their negative predictive values were 81.2% and 78.6%; and the global values were 78.8% and 74.3%. Thus, the most accurate test to distinguish an LSIL from an HSIL was the association of a suspect DNA profile and the presence of MIB-1- or ICBP90-positive cells in the upper two thirds of the epithelium.

Antibodies, Antinuclear↗
PubMed1991

Calcium channels in PDGF-stimulated A172 cells open after intracellular calcium release and are not voltage-dependent.

Using laser image cytometry and Indo-1 fluorescence, we investigated the intracellular free Ca2+ concentration ([Ca2+]i) of confluent A172 human glioblastoma cells stimulated by the BB homodimer of platelet-derived growth factor (PDGF-BB). The shape of the calcium transients and the delay time between stimulation and the beginning of the transient varied considerably. The percentage of responsive cells, the peak [Ca2+]i and the duration of the response were directly related to PDGF-BB dose, while the delay time was inversely related; the maximal response occurred at a PDGF-BB concentration of 20 ng/ml. Studies with EGTA and inorganic calcium-channel blockers (Ni2+, La3+) showed that the increase of [Ca2+]i resulted from initial release of intracellular stores and subsequent calcium influx across the plasma membrane. Opening of calcium channels in the plasma membrane, monitored directly by studying Mn2+ quenching of Indo-1 fluorescence, was stimulated by PDGF-BB and blocked by La3+; the opening occurred 55 +/- 10 s after the initial increase in [Ca2+]i. Therefore, in these tumor cells, intracellular release always occurs before channel opening in the plasma membrane. Depolarization of cells with high extracellular [K+] did not generally induce calcium transients but did decrease calcium influx. L-type calcium-channel blockers (verapamil, nifedipine, and diltiazem) had little or no effect on the calcium influx induced by PDGF-BB. These results indicate that PDGF-BB induces calcium influx by a mechanism independent of voltage-sensitive calcium channels in A172 human glioblastoma cells.

Calcium↗
PubMed1998

Congenital melanocytic nevi with placental infiltration by melanocytes: a benign condition that mimics metastatic melanoma.

BACKGROUND: Placental metastases from cutaneous malignant melanoma from both the mother and the fetus have been reported. The finding of benign-appearing melanocytes in the placenta in association with congenital melanocytic nevi (CMN) is more exceptional, with only 6 reports in the literature. Clinically, the finding of melanocytes in the placenta in this setting can be alarming and might erroneously lead to the diagnosis of metastatic melanoma. OBSERVATIONS: Herein, we describe 3 additional patients with CMN with placental infiltration by melanocytes with a benign phenotype. In the results of immunoperoxidase stains, the melanocytic cells were positive for S-100 protein and HMB-45 in the 2 lesions available for study. Staining of placental vessels with Ulex europaeus agglutinin I (Vector Laboratories, Burlingame, Calif) failed to show intravascular melanocytes in the 1 lesion available for study. We report for the first time DNA diploidy in 2 lesions available for study, which were analyzed by DNA image cytometry. We describe the first patient with a relatively small, nongiant CMN. CONCLUSIONS: We support the notion of the aberrant migration of melanocytes from the neural crest during fetal development as the most likely explanation of this phenomenon and note the similarity to the association of CMN and leptomeningeal melanocytosis. However, the precise histogenesis of this process remains uncertain. Most importantly, our data provide further evidence for the benign nature of this condition. Awareness of this entity is of vital importance in avoiding overdiagnosis of melanoma in this clinical setting.

Adolescent↗
PubMed1996

In vitro characterization of prolactin-induced effects on proliferation in the neoplastic LNCaP, DU145, and PC3 models of the human prostate.

BACKGROUND: Proliferation of normal and tumoral prostate tissue is regulated by androgens and various growth factors. We characterized the in vitro proliferative influence of prolactin (PRL) in androgen-sensitive and androgen-insensitive human prostate cancers. METHODS: The biologic models employed included the androgen-sensitive LNCaP and the androgen-insensitive DU145 and PC3 cell lines. PRL-induced influences (0.1-10 mIU/ml of medium) on proliferation were assessed using the colorimetric methylthiotetrazole assay. Androgen sensitivity in the three cell lines was determined by assessing the proliferative influence of dihydrotestosterone (DHT) (0.1-10 nM). PRL-induced modifications in PC3 cell kinetics were assessed using Feulgen-stained nuclear image cytometry. RESULTS: Although DHT markedly stimulated LNCaP proliferation, it had no proliferative effect on the DU145 and PC3 cell lines. By contrast, PRL significantly modulated the proliferation of the DU145 and PC3 lines, but exerted weak, if any, effect on the proliferation of the LNCaP cell line. PRL increased the percentage of PC3 proliferating cells (i.e., cells in the S/G2 phases of the cell cycle) at low doses (0.1 mIU/mL) and decreased this percentage at high doses (10 mIU/ml). CONCLUSIONS: Proliferation of androgen-insensitive human prostate cell lines can be significantly modulated by prolactin.

Androgens↗
PubMed2000

Squamous papilloma of the urinary tract is unrelated to condyloma acuminata.

BACKGROUND: To the authors' knowledge, there is no previous report of squamous papilloma of the urinary tract. It is uncertain whether there is a correlation between squamous papilloma, condyloma acuminatum, and verrucous carcinoma. METHODS: The authors evaluated the clinical and pathologic features of squamous papilloma (5 of the bladder, 2 of the urethra), condyloma acuminatum (3 cases), and verrucous carcinoma (3 cases) of the urinary bladder and performed human papillomavirus (HPV) DNA in situ hybridization studies to determine whether HPV was a common feature shared by these lesions. In addition, DNA ploidy evaluation by image cytometry and p53 immunohistochemical staining were performed. RESULTS: Squamous papilloma of the urinary tract occurred in elderly women and followed a benign clinical course with infrequent recurrence. All squamous papillomas were HPV DNA negative and DNA diploid with no or minimal p53 nuclear accumulation. Condyloma acuminata of the bladder contained HPV DNA, increased p53 protein expression, and aneuploid DNA content. All three cases of condyloma acuminata were associated with coexistent condylomata of the external genitalia, and two required pelvic exenteration for uncontrolled expansile growth. Verrucous carcinoma of the bladder occurred in elderly patients. All three cases of verrucous carcinoma were negative for HPV DNA and DNA aneuploid, and they exhibited consistent p53 expression. CONCLUSIONS: These data indicate that squamous papilloma is a distinct entity not related to condyloma or verrucous carcinoma. These lesions are benign, HPV DNA negative, DNA diploid, and they lack p53 overaccumulation.

Adult↗
PubMed1998

Efficient, interactive, and three-dimensional segmentation of cell nuclei in thick tissue sections.

Segmentation of intact cell nuclei in three-dimensional (3D) images of thick tissue sections is an important basic capability necessary for many biological research studies. Because automatic algorithms do not correctly segment all nuclei in tissue sections, interactive algorithms may be preferable for some applications. Existing interactive segmentation algorithms require the analyst to draw a border around the nucleus under consideration in all successive two-dimensional (2D) planes of the 3D image. The present paper describes an algorithm with two main advantages over the existing method. First, the analyst draws borders only in 2D planes that cut approximately through the center of the nucleus under consideration so that the nuclear borders generally are most distinct. Second, the analyst draws only five borders around each nucleus, and then the algorithm interpolates the entire surface. The algorithm results in segmented objects that correspond to individual, visually identifiable nuclei. The segmented surfaces, however, may not exactly represent the true nuclear surface. An optional, automatic surface optimization algorithm can be applied to reduce this error.

Algorithms↗
PubMed1998

Automated image analysis system for detecting boundaries of live prostate cancer cells.

Image analysis provides a powerful tool for quantifying cell motility and has been used to correlate motility with metastatic potential in an animal model of prostate cancer. However, widespread use of this image analysis method has been limited because earlier methods of quantitative analysis required time-intensive and subjective manual tracing of cell contours. In this report, we describe a fully automated image segmentation algorithm for detection and morphometric description of prostatic cells. The segmentation system was tested on prostate cell images generated from Hoffman modulation contrast microscopy (47 cells at 64 time points = 3,008 images) and differential interference contrast microscopy (29 cells at 64 times points plus 1 cell at 62 time points = 1,918 images). Morphometric measurements were derived from computer-determined cell boundaries and compared with the same measurements derived from manually traced cell boundaries. Final correlation coefficients for area and perimeter measurements for Hoffman and differential interference contrast microscopy were (0.76, 0.62) and (0.93, 0.93), respectively. Results with our differential interference contrast images demonstrate that our segmentation algorithm reliably and efficiently replaces the need for manually traced cell boundaries in addition to eliminating intraobserver variation. Our automated segmentation process will have immediate utility in our motility analysis system that relates cell motility with metastatic potential of prostate cancer.

Adenocarcinoma↗
PubMed1998

Image analysis enhancement of the laser scanning cytometer.

The laser scanning cytometer offers a range of novel applications and the capacity for direct visual validation of experiments through sample analysis on a microscope slide. Linkage of the instrument to an image analysis system through standard connections and software enhances the capabilities of the instrument in image capture and manipulation. In this technical note, we describe a simple linkage between the LSC and the Kontron KS100 Image Analysis System, an example of a standard commercial image processing instrument.

Image Cytometry↗
PubMed1999

A new multiparameter flow cytometer: optical and electrical cell analysis in combination with video microscopy in flow.

BACKGROUND: Flow cytometers, which are commercially available, do not necessarily meet all demands of actual biomedical research. This is the case for the investigation of mechanisms involved in cell volume regulation, which requires electrical volume measurement and ratiometric multichannel fluorescence analysis for the simultaneous assessment of different physiologic parameters (intracellular pH and the intracellular concentration of calcium ions, etc). METHODS AND RESULTS: We describe the construction of a new nonsorting flow cytometer designed for the simultaneous acquisition of seven parameters including fluorescence signals, forward and perpendicular light scatter, cell volume according to the electrical Coulter principle, and flow cytometric imaging. The instrument is equipped with three different light sources. A tunable argon-ion laser generates efficient excitation of the most standard fluorescent probes in the visible spectral range, and an arc lamp provides the means for ultraviolet excitation at low cost. Because of the spatial filtering by the excitation and detection optics, two independent sets of dual fluorescence measurements can be performed, a prerequisite for flexible ratiometric fluorescence analysis. A flow video microscope integrated into the optical system optionally generates either brightfield or phase images of selected flowing particles. Only particles whose individual datasets meet predefined gating conditions are imaged in real time. To avoid smear effects, the motion of the object to be imaged (speed approximately 8 m/s) is frozen on the target of a CCD camera by flash illumination. For this purpose, a high radiance gas discharge lamp with 25-mJ electric pulse energy provides an illumination time of 18 ns (full width half maximum). Test results obtained from latex spheres and cells are shown. CONCLUSIONS: Test results indicate that our instrument can perform Coulter measurements in combination with flexible optical analysis. Moreover, integration of an adapted video microscope into a flow cytometer is an approach to overcome the gap between flow and image cytometry.

Animals↗
PubMed1999

Artificial neural network-aided image analysis system for cell counting.

BACKGROUND: In histological preparations containing debris and synthetic materials, it is difficult to automate cell counting using standard image analysis tools, i.e., systems that rely on boundary contours, histogram thresholding, etc. In an attempt to mimic manual cell recognition, an automated cell counter was constructed using a combination of artificial intelligence and standard image analysis methods. METHODS: Artificial neural network (ANN) methods were applied on digitized microscopy fields without pre-ANN feature extraction. A three-layer feed-forward network with extensive weight sharing in the first hidden layer was employed and trained on 1,830 examples using the error back-propagation algorithm on a Power Macintosh 7300/180 desktop computer. The optimal number of hidden neurons was determined and the trained system was validated by comparison with blinded human counts. System performance at 50x and lO0x magnification was evaluated. RESULTS: The correlation index at 100x magnification neared person-to-person variability, while 50x magnification was not useful. The system was approximately six times faster than an experienced human. CONCLUSIONS: ANN-based automated cell counting in noisy histological preparations is feasible. Consistent histology and computer power are crucial for system performance. The system provides several benefits, such as speed of analysis and consistency, and frees up personnel for other tasks.

Cell Count↗
PubMed1999

In vitro motility evaluation of aggregated cancer cells by means of automatic image processing.

BACKGROUND: Set up of an automatic image processing based method that enables the motility of in vitro aggregated cells to be evaluated for a number of hours. METHODS: Our biological model included the PC-3 human prostate cancer cell line growing as a monolayer on the bottom of Falcon plastic dishes containing conventional culture media. Our equipment consisted of an incubator, an inverted phase contrast microscope, a Charge Coupled Device (CCD) video camera, and a computer equipped with an image processing software developed in our laboratory. This computer-assisted microscope analysis of aggregated cells enables global cluster motility to be evaluated. This analysis also enables the trajectory of each cell to be isolated and parametrized within a given cluster or, indeed, the trajectories of individual cells outside a cluster. RESULTS: The results show that motility inside a PC-3 cluster is not restricted to slight motion due to cluster expansion, but rather consists of a marked cell movement within the cluster. CONCLUSIONS: The proposed equipment enables in vitro aggregated cell motility to be studied. This method can, therefore, be used in pharmacological studies in order to select anti-motility related compounds. The compounds selected by the equipment described could then be tested in vivo as potential anti-metastatic.

Algorithms↗