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PubMed1996

Basaloid-squamous carcinoma of the esophagus. A clinicopathologic, DNA ploidy, and immunohistochemical study of seven cases.

Basaloid-squamous carcinoma (BSC) of the esophagus is a rare but interesting neoplasm that occurs primarily in the upper aerodigestive tract. In this study, we reviewed 371 cases of esophageal malignancies and detected seven cases (1.9%) of BSC. The clinicopathologic features, light and electron microscopic findings, and immunohistochemical localization of various differentiation-related antigens, including cytokeratin (CK) subtypes, p53, and epidermal growth factor receptor (EGFR), were examined. DNA ploidy was also determined in an effort to characterize the biologic features of these tumors. The tumors were classified as stage I (n = 1), IIB (n = 3), III (n = 2) or IV (n = 1). Six patients had lymph node metastasis, in four the metastatic carcinoma exhibited basaloid components. Histologically, all the tumors displayed a biphasic pattern of basaloid and squamous components. The former predominated in three cases, the latter in four cases. All the tumors contained solid growth of basaloid cells with microcystic patterns and stromal hyalinosis as well as palisading of cells. Ultrastructurally, markedly replicated basement membrane was observed. Immunohistochemistry revealed staining with only CK 14 and CK 19 antibodies in the periphery of the basaloid tumor nests. These antibodies were also positive in the basal layer of normal esophagus. Diffuse immunoreactivity for EGFR was demonstrated in all the tumors. Five tumors displayed p53 nuclear immunoreactivity. All of the basaloid components demonstrated aneuploidy by DNA image cytometry. We conclude that BSC is a distinct type of esophageal carcinoma that should be differentiated from the usual types of esophageal carcinoma and may be associated with aggressive biologic behavior.

Aged↗
PubMed1996

DNA ploidy analysis of mucinous cystic tumors of the pancreas. Correlation of aneuploidy with malignancy and poor prognosis.

BACKGROUND: Benign and malignant pancreatic mucinous tumors differ in proliferative activity, production of tumor markers, and expression of growth factors. DNA ploidy is a useful index of aggressiveness and poor survival in ductal adenocarcinoma, but has not been studied in pancreatic cystic tumors. METHODS: The DNA ploidy status of Fuelgen-stained tissue sections of pancreatic mucinous cystic tumors was evaluated by image cytometry and related to clinical outcome obtained by case record review. RESULTS: Ploidy status correlated with malignancy and poor clinical outcome. All benign mucinous cystadenomas (n = 13) were diploid and cured by resection. Patients with diploid cystadenocarcinomas (n = 6) had an 83% survival rate following resection, while patients with aneuploid cystadenocarcinomas (n = 5) all died (four of disease and the fifth of another cause). The difference in survival between the diploid and aneuploid carcinomas was significant (P = 0.04). CONCLUSIONS: DNA ploidy status of pancreatic mucinous tumors by image analysis provides quantifiable information that may be predictive of clinical outcome. DNA aneuploidy appears to be a significant differential factor in pancreatic mucinous tumors and is associated with shortened survival in mucinous cystadenocarcinomas.

Adult↗
PubMed1998

Application of fluorescence resonance energy transfer in the clinical laboratory: routine and research.

Fluorescence resonance energy transfer (FRET) phenomenon has been applied to a variety of scientific challenges in the past. The potential utility of this biophysical tool will be revisited in the 21st century. The rapid digital signal processing in conjunction with personal computers and the wide use of multicolor laser technology in clinical flow cytometry opened an opportunity for multiplexed assay systems. The concept is very simple. Color-coded microspheres are used as solid-phase matrix for the detection of fluorescent labeled molecules. It is the homogeneous assay methodology in which solid-phase particles behave similarly to the dynamics of a liquid environment. This approach offers a rapid cost-effective technology that harnesses a wide variety of fluorochromes and lasers. With this microsphere technology, the potential applications for clinical flow cytometry in the future are enormous. This new approach of well-established clinically proven methods sets the stage to briefly review the theoretical and practical aspects of FRET technology. The review shows various applications of FRET in research and clinical laboratories. Combination of FRET with monoclonal antibodies resulted in a boom of structural analysis of proteins in solutions and also in biological membranes. Cell surface mapping of cluster of differentiation molecules on immunocompetent cells has gained more and more interest in the last decade. Several examples for biological applications are discussed in detail. FRET can also be used to improve the spectral characteristics of fluorescent dyes and dye combinations, such as the tandem dyes in flow and image cytometry and the FRET primers in DNA sequencing and polymerase chain reactions. The advantages and disadvantages of donor-acceptor dye combinations are evaluated. In addition, the sensitivity of FRET provides the basis for establishing fast, robust, and accurate enzyme assays and immunoassays. Benefits and limitations of FRET-based assays are thoroughly scrutinized. At the end of the paper we review the future of FRET methodology.

Diagnostic Tests, Routine↗
PubMed2005

Cytologic and DNA-cytometric follow-up of oral leukoplakia after CO2- and Er:YAG-laser assisted ablation: a pilot study.

BACKGROUND AND OBJECTIVES: The aim of the present pilot study was to determine therapeutic responses to Er:YAG- and CO(2)-laser ablation in patients with oral leukoplakia as evaluated by means of exfoliative cytology (EC) and DNA-image-cytometry (DNA-I). STUDY DESIGN/MATERIALS AND METHODS: Ten patients exhibiting a total of 16 lesions affecting a variety of intraoral sites were randomly treated with either (1) an Er:YAG laser (300 mJ/pulse, 10 Hz, defocused mode) (ERL), or (2) an CO(2) laser (4-6 W, 20-50 Hz, focused mode) (CO). Brush (B) and incisional (I) biopsies were obtained from the respective lesions immediately before treatment (B, I) as well as 24-96 weeks postoperatively (B). In cases, in which EC revealed suspicious cells, nuclear DNA-contents were measured using a TV image analysis system. RESULTS: Both treatment approaches resulted in a complete (C) or partial (P) remission of all investigated lesions. In particular, ERL exhibited C(3), P(5), and CO C(5), P(3). However, in the CO group, two of eight lesions showed a recurrence 32-48 weeks following treatment. Among all investigated lesions, both histological and EC/DNA-I diagnosis revealed no sign of malignancy or dysplasia before or following laser assisted ablation. CONCLUSIONS: Within the limits of the present study, it may be concluded that both treatment approaches seem to have limitations to achieve predictable eradication of oral leukoplakia.

Adult↗
PubMed1998

Retrospective evaluation of DNA ploidy of hepatocarcinoma on cytologic samples.

DNA ploidy was evaluated by image cytometry in a series of 84 hepatocellular carcinomas diagnosed by fine-needle aspiration biopsy. In the series were included eight cases originally diagnosed as suspect and reclassified as well-differentiated hepatocarcinoma. The study was retrospectively performed on Papanicolaou-destained, Feulgen-restained smears. The 5c exceeding rate and the visual interpretation of the corresponding histograms were evaluated and compared with size of the tumors, serum alpha-fetoprotein values, hepatic functional staging, and patient survival. Sixty-eight cases were aneuploid and 16 euploid (9 diploid and 7 polyploid). Four of the eight cytologically suspect cases were aneuploid. Statistical analysis showed an association between size and cytologic grading, 5c exceeding rate and cytologic grading, and between aneuploidy and multiple tumors; in a Cox multivariate DNA content analysis, aneuploidy and multiple tumors were the two prognostically significant variables. DNA ploidy evaluation by static cytometry of hepatic tumors may be useful in the diagnosis on cytologic samples and could represent an independent prognostic parameter in predicting the survival outcome of patients with hepatocellular carcinoma.

Adult↗
PubMed2006

Verrucous carcinoma of the buccal mucosa: histopathological, cytological and DNA-cytometric features.

We describe a patient with an exophytic oral lesion diagnosed as verrucous carcinoma. The lesion existed without metastases, at least 5 years. Local excisions led to recurrences and continuous expansion. Scalpel biopsies for histopathological and polymerase chain reaction examination were obtained from characteristic regions of the lesion. Brush biopsies for exfoliative cytology (EC) were taken, in order to screen the mucosal area covered by the lesion. After Feulgen restaining of the smears, nuclear DNA contents were measured using a TV image analysis system. An exophytic lesion of the buccal mucosa was diagnosed as low-grade malignant through histopathology and EC combined with DNA-image cytometry (peritetraploid DNA-aneuploidy). Due to almost normal microscopic appearance of the epithelium of verrucous carcinoma, thorough cytological/DNA-cytometric and histological examinations are needed. Brush biopsies of such neoplastic oral lesions showing DNA-aneuploidy with peritetraploid stemlines should be used for diagnosis and follow-up examination of these patients.

Aged↗
PubMed2001

Automated nuclear image morphometry on fine needle aspiration smears of malignant round cell tumors.

OBJECTIVE: To analyze nuclear image morphometry in fine needle aspiration cytology smears of different groups of malignant round cell tumors (MRCTs) to evaluate its diagnostic role. STUDY DESIGN: In this study there were 55 cases of MRCT, consisting of 18 Ewing's sarcoma (EW), 10 neuroblastoma (NB), 5 non-Hodgkin's lymphoma (NHL), 6 rhabdomyosarcoma (RMS), 4 peripheral neuroectodermal tumor (PNET), 8 Wilm's tumor (WT), 2 retinoblastoma (RB) and 2 undifferentiated round cell tumor (URCT). A Leica image cytometer with Quantimet 600 software (Leica, Cambridge, U.K) was used to measure nuclear area, nuclear diameter, nuclear perimeter, nuclear convex perimeter (CP), nuclear roundness and nuclear convex area on hematoxylin and eosin-stained cytologic smears. At least 100 cells were studied in each case. RESULTS: The RB group of tumors showed the highest mean nuclear area (NA), convex area (CA), CP, diameter (D), perimeter (P) and roundness (R). RMS had the highest mean CA, and URCT had the highest mean roundness. ANOVA was performed on the tumors and showed significant differences for all the variables in all the groups (P < .000). All the morphometric data (except roundness) were significantly different in RMS versus all other MRCTs except RB. Similarly, morphometric data on WT were also significantly different from that on NHL. Most of the morphometric data (except CA and R) showed significant differences between RB and all other MRCTs except RMS. PNET, EW and NB could not be differentiated with those variables. CONCLUSION: RMS and RB could successfully be differentiated from all other MRCTs with the help of morphometry. It was not possible to differentiate RMS and RB by image cytometry (ICM) since the ICM data overlapped in those two groups. It was possible to differentiate WT and NHL with ICM. Nuclear ICM was not significantly different in the NB, PNET and ES groups, and probably ICM would not be very helpful to differentiate these groups of MRCT.

Biopsy, Needle↗
PubMedDate not supplied

DNA quantification as prognostic factor in a case of acinar cell carcinoma of the parotid gland, diagnosed by FNA.

Hereby we present a case of a 43-years-old male who complained of a three years history preauricular painful mass. Fine needle aspiration cytology was performed, diagnosing of compatible with acinar cell carcinoma, thus DNA quantification by image cytometry was carried out. Biological parameters studied (ploidy, S-phase, 5-c exceeding rate) showed that it is a low grade of malignancy lesion. Total parotidectomy conservative of facial nerve was recommended, without regional lymphadenectomy. Patient remains, one year later, asymptomatic and free of disease.

Adult↗
PubMed1996

DNA quantification as a prognostic factor in prostatic adenocarcinoma.

OBJECTIVE: To determine if DNA quantification on fine needle aspiration (FNA) has a predictive value in cancer of the prostate. STUDY DESIGN: Fifty-four patients with cancer of the prostate during the years 1979-1989 were selected; all were diagnosed by FNA. The smears were studied using image cytometry. RESULTS: With the type of histogram and value of entropy, two large groups with high and low degrees of malignancy were obtained. Survival was utilized as a variable of interest. Kaplan-Meier survival curves for both groups were formulated, and the results were supported with statistical data. CONCLUSION: The survival differences between both groups were statistically significant (P < .001), thus demonstrating the predictive value of DNA quantification.

Adenocarcinoma↗
PubMed1998

[Studies on DNA content in sperm of men with oligozoospermia].

The aim of our study was to analysis the ploidy of spermatozoa from oligozoospermic individuals. The study was performed in 48 patients with oligozoospermia. Spermatozoal DNA was analysed using MagiCal imaging cytometry. The percentage of aneuploid cells was determined using CYT-2 program. The results indicated aneuploidy in 73% of the examined patients. The amount of cells with abnormal DNA content ranged from 30 to 100%. Monitoring of the DNA in human spermatozoa may have practical applications in establishing the reasons for infertility as well as for screening of the semen for assisted reproduction techniques.

Aneuploidy↗
PubMed2001

Correlation between silver-stained nucleolar organizer region area and cell cycle time.

BACKGROUND: The relationship between the population doubling time and the quantity of silver-stained nucleolar organizer region (AgNOR) interphase proteins was studied in cell culture at three different temperatures used to modulate the cell cycle duration. METHODS: After MIB 1 and AgNOR combined staining, the quantity of AgNOR proteins was measured in cycling cells by image cytometry. RESULTS: Among the several parameters calculated, the AgNOR relative area showed a strong correlation with the changes of the population doubling time induced by different temperatures. CONCLUSIONS: The results support the hypothesis that the cell cycle time and the size of the ribogenesis machinery are coregulated and that measurements of AgNORs can thus be used as a static evaluation of the cell cycle duration in arbitrary units.

Antigens, Nuclear↗
PubMed2004

Evaluation of genomic changes in a large series of malignant ovarian germ cell tumors--relation to clinicopathologic variables.

Malignant ovarian germ cell tumors (mOGCT) affect women in their reproductive years, making fertility-saving treatment important. A reliable prediction of the clinical behavior is essential for an optimal therapeutic approach. The genetic changes and molecular mechanisms underlying these rare tumors remain poorly understood. To address these issues, we performed DNA ploidy analysis by high-resolution image cytometry in a series of 47 mOGCT and correlated the findings with the DNA copy number changes detected by comparative genomic hybridization (CGH) and clinical outcome. Of 47 tumors, 15 were diploid, 14 were tetraploid, 2 were polyploid, and 13 were aneuploid. All the immature teratomas were diploid, in contrast to the dysgerminomas and endodermal sinus tumors. The International Federation of Gynecology and Obstetrics (FIGO) staging, residual tumors after surgery, and DNA ploidy distribution were significant, independent prognostic factors in survival analysis. The study revealed that the number of DNA copy number aberrations was increased in tetraploid and aneuploid tumors as compared to diploid tumors. Furthermore, a high percentage of aneuploid nuclei in a sample were associated with a complex CGH profile of the tumor in question. The present study confirms that DNA aneuploidy assessment by image analysis may be linked to genetic instability, which is detected as genetic aberrations by CGH. DNA ploidy gives significant prognostic information in addition to the clinical stage in mOGCT with FIGO stage II-IV.

Adolescent↗
PubMed2004

[Correlation of cytologic and DNA ploidy evidence and prognosis in childhood Wilm's tumor].

A retrospective evaluation of the clinical features, cytological, biological (DNA) and histological data on 29 children with Wilms' tumor. Two prognosis-related groups were identified: (1) good outcome (17)--all children, aged 4-15, had survived by end of follow-up; (2) bad outcome (12)--all children died within 4 months--4 years. Cytological examination by fine-needle biopsy of smears was carried out prior to preoperative neoadjuvant therapy in all 29 patients; in addition, print smears from resected tumor were taken from 21. Blastemal, stromal, epithelial and anaplastic cells levels were estimated by the semi-quantitative procedure (0, 1+, 2+, 3+). DNA ploidy was determined by image cytometry after destaining slides and re-staining them according to Feulgen. It was shown that favorable outcome could be expected when fine-needle biopsy of print smears identified DNA diploidy and marked drug-related pathomorphosis involving fewer blastemal cells and signs of cell dystrophy and tumor necrosis. When anaplastic and aneuploid cells were detected, matched by the absence of therapeutic pathomorphosis, outcome was poor.

Adolescent↗
PubMed2000

Bcl-2: bax and bcl-2: Bcl-x ratios by image cytometric quantitation of immunohistochemical expression in ovarian carcinoma: correlation with prognosis.

Bcl-2 is a proto-oncogene which is involved in prolonging cell survival by inhibiting programmed cell death. Bax and bcl-x are members of the bcl-2 family; when overexpressed, they can counteract the ability of bcl-2 to inhibit apoptosis. This suggests a model in which the ratios of bcl-2 to bax and bcl-x can be used to determine response to therapy and prognosis. The expression of bcl-2, bax and bcl-x was studied in 50 ovarian carcinomas. The percentage of positive area immunostained (PPA) in the nucleus and cytoplasm of each ovarian carcinoma was quantitated in 15 high power fields by image cytometry. The ratios were obtained by dividing the PPA of bcl-2 by the PPA of bax and bcl-x. 17 of 50 ovarian carcinomas (34%) stained positively for bcl-2, 39 for bax (78%) and 47 for bcl-x (94%). Although there is no significant statistical correlation between expression of bcl-2, bax or bcl-x and grade (P = 0.15; P = 0. 47; P = 0.56), stage (P = 0.71; P = 0.6; P = 0.42), and overall or disease-free survival (P = 0.26; P = 0.55; P = 0.16), increased bcl-2 expression was demonstrated in patients with shortened overall and disease-free survival. Also, increased expression of bax and bcl-x was associated with increased overall and disease-free survival. Bcl-2:bax and bcl-2:bcl-x ratios less than 1 are associated with survival advantage, although not statistically significant (P = 0.83; P = 0.93). Image cytometric measurement of bcl-2, bax, and bcl-x expression is feasible. There is a tendency for their expression to correlate with prognosis in ovarian carcinomas.

Carcinoma↗
PubMed2003

Nuclear texture and chromatin structure in OV1/VCR human multidrug-resistant cell line.

Previous studies have demonstrated that multidrug-resistant leukemic cells displayed nuclear texture changes. In this work, the human ovarian carcinoma cell line IGROV1 and its multidrug-resistant variant OV1/VCR were studied. Cell smears of these cell populations were analysed by image cytometry. As compared to sensitive cells, OV1/VCR display a chromatin global decondensation as assessed by textural features analysis. In order to correlate this decondensation with alterations in chromatin structure, DNase I was used. OV1/VCR DNA displayed an increased DNase I sensitivity, suggesting an increased chromatin accessibility. Furthermore, OV1/VCR cells displayed an increased level in acetylated histone H4, a mechanism known to be associated with transcriptionally active chromatin and relaxed chromatin conformation.

Cell Line, Tumor↗
PubMed2005

Regenerative and predictive medicine of cardiovascular disease: the 9th Leipziger Workshop and the 2nd International Workshop on slide based cytometry.

Slide-based cytometry (SBC) and related techniques offer unique tools to perform complex immunophenotyping, thereby enabling diagnostic procedures at very early disease stages. Multicolor or polychromatic analysis of cells by SBC is of special importance, not only as a cytomics technology platform but also for patients with low blood volume such as neonates. The exact knowledge of the location of each cell on the slide allows restaining and subsequent reanalysis of the specimen. These separate measurements of the same specimen can be fused to one data file (merging), thus increasing the information obtained per cell. Relocalization and optical evaluation of the cells, a feature typical of SBC, can be of integral importance for cytometric analysis. Due to this feature, artifacts can be excluded and morphology of measured cells can be documented. Predictive medicine aims at the detection of changes in patient's state before the manifestation of the disease or its complications. Such instances concern multiorgan failure in sepsis or noninfectious posttraumatic shock in intensive care patients or the pretherapeutic identification of high-risk patients undergoing cancer cytostatic therapy. Early anti-infectious or antishock therapy and curative chemotherapy in combination with stem cell transplantation may provide better chances of patients' survival at concomitant cost containment. Predictive medicine that guides early individualized decrease or cessation of therapy may lower or abrogate potential therapeutic side effects (individualized medicine). Regenerative medicine concerns patients who have diseased and injured organs and may be treated with transplanted organs. However, there is a severe shortage of donor organs that is worsening yearly given the aging population. Regenerative medicine and tissue engineering apply the principles of cell transplantation, material science, and bioengineering to construct biological substitutes that will restore and maintain normal function in diseased and injured tissues. Neovascularization is promoted by bone marrow-derived endothelial progenitor cells that lead to the formation of entirely new vessels into ischemic tissue. With this knowledge, many therapeutical borders can be skipped. Diseases formerly uncontrolled can be corrected with stem cells to provide causal healing with regeneration processes. The 9th Leipziger Workshop combined with the 2nd International Workshop on SBC aimed to offer new methods in image cytometry and SBC for solutions in clinical research. It moved toward practical applications in clinics and the clinical laboratory. This development will be continued in 2005 at the upcoming Leipziger Workshop and the 3rd International Workshop on SBC.

Cardiovascular Diseases↗
PubMed1998

Microvascular cytomegalovirus endothelialitis of the lung: a possible cause of secondary pulmonary hypertension in a patient with AIDS.

Postmortem examination of the lungs of a patient with advanced AIDS who had developed pulmonary arterial hypertension late in the course of the illness demonstrated extensive cytomegalovirus (CMV) infection in endothelial cells of the lung microvasculature. Enlarged CMV-infected endothelial cells were present in virtually all histologic sections of the lungs, protruded into and compromised the lumens of the small vessels they lined, and were estimated by image cytometry of immunohistochemically stained sections to comprise 0.8% of the total lung tissue volume. Comparison with experimental microvascular embolization studies suggests that this amount of compromise of the microvascular luminal area of the lung is sufficient to elevate pulmonary arterial pressure significantly. Pathologic features in this case differed from both the plexogenic arteriopathy seen in previously reported cases of AIDS-associated primary pulmonary hypertension and the usual form of CMV pneumonitis in AIDS in which alveolar epithelial cells are the predominant site of infection.

AIDS-Related Opportunistic Infections↗
PubMed1996

Cell cycle expression of steroid receptors determined by image analysis on human breast cancer cell line: a hypothesis on the effects of antiestrogens.

Tamoxifen is the widespread anti-hormonal compound used for the treatment of human breast cancer. It is admitted that its effects are mediated via estrogen receptors (ER) but the molecular basis of its activity has yet to be clearly defined. In this work, we have developed a new image cytometry procedure in order to clarify the interactions between steroid receptors and tamoxifen at the cell cycle kinetic level. On untreated cells, an increase of ER level and a decrease of progesterone receptor (PR) level during the G0/G1 phase were demonstrated. Then, the ER and PR levels fell during the S-phase until the beginning of G2/M phase, where an increase was observed, especially for PR. These results suggest that ER is synthesized preferentially during the G0/G1 transition and PR during the S/G2 transition. After short-term tamoxifen treatment an augmentation of ER level was observed which was not dose-dependent, suggesting an increase in receptor translation rather than an augmentation of ER synthesis. PR level declined in the majority of the population leading to a selection of a subset of proliferating PR negative cells after treatment. These data demonstrate that the synthesis of steroid receptors is linked with the progression of cells through the cell cycle and indicate that tamoxifen blocks MCF-7 cells in G1 via its interactions with ER. Our multifluorescence imaging procedure appears to provide a rapid and quantitative approach which is useful for investigating alterations in steroid receptors after endocrine treatment.

Antineoplastic Agents, Hormonal↗