1997 ESACP consensus report on diagnostic DNA image cytometry. Part II: Specific recommendations for quality assurance. European Society for Analytical Cellular Pathology.
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Humans↗
Biomedical subjects
A Böcking
Publications and source records attributed to A Böcking.
1997 ESACP consensus report on diagnostic DNA image cytometry. Part I: basic considerations and recommendations for preparation, measurement and interpretation. European Society for Analytical Cellular Pathology.
A task force of invited experts in the field of diagnostic DNA image cytometry, especially consisting of participants from the PRESS (Prototype Reference Standard Slides) and EUROPATH (European Pathology Assisted by Telematics for Healthcare) projects, but open to any other scientist or physician revealing experience in that new diagnostic procedure (names are given in the Annex A) agreed upon the following updated consensus report during the 5th International Congress of the ESACP 1997 in Oslo. This report is based on the preceeding one [9] and on results of the above mentioned European research projects. It deals with the following items: Biological background and aims of DNA image cytometry, Principles of the method, Basic performance standards, Diagnostic interpretation of DNA measurements, Recommendations for practical use.
Aneuploidy↗
Atomic force microscopy in effusion cytology.
OBJECTIVE: To investigate whether atomic force microscopy (AFM) in combination with classical light microscopy allows simple identification of surface structures of cells from pleural and ascitic fluids for diagnostic purposes in place of scanning electron microscopy (SEM). STUDY DESIGN: We examined a total of 180 cells obtained from 9 reactive pleural or peritoneal effusions, 14 associated with carcinomatosis from histologically confirmed tumors and 5 from mesotheliomas. Cells of interest were selected in air-dried, uncovered, May-Grünwald-Giemsa (MGG)-stained smears and subsequently investigated by AFM. Incorporation of a very compact AFM scanner into the nose piece of a conventional Axioscope light microscope allowed alternating application of both techniques. RESULTS: AFM was able to detect cell surface structures, such as microvilli, phagocytic pits, secretory blebs and lytic holes. The image resolution was sufficient but not as good as that with SEM. We found differences in number, length and diameter of microvilli between cells from mesotheliomas and from metastatic adenocarcinomas. CONCLUSION: As AFM can be carried out in combination with light microscopy quickly and easily on uncovered, MGG-stained smears, we propose this method as a suitable tool for obtaining additional useful information in routine cytologic diagnosis of effusions.
Adenocarcinoma↗
Static DNA cytometry as a diagnostic aid in effusion cytology: I. DNA aneuploidy for identification and differentiation of primary and secondary tumors of the serous membranes.
OBJECTIVE: To determine whether DNA aneuploidy is a sensitive and specific marker for the identification of tumor cells in effusions and whether the pattern of DNA aneuploidy can provide important information for the differential diagnosis of primary and secondary tumors of the serous membranes. STUDY DESIGN: One hundred eight malignant mesotheliomas as well as 102 metastatic carcinomas of the serous membranes were obtained from routine cytologic and histologic material. One hundred reactive effusions were investigated as controls. Nuclear DNA contents were measured after Feulgen staining using a TV image analysis system. RESULTS: DNA aneuploidy was assumed if abnormal DNA stemlines, a coefficient of variation of the first DNA stemline > or = 10%, or cells > 9c were observed. On this basis the prevalence of DNA aneuploidy in mesotheliomas was 83% for cytologic and 84% for histologic material. In effusions of metastatic carcinomas it was 100%. None of the 100 reactive effusions revealed DNA aneuploidy (prevalence, 0%). Positive predictive value for mesotheliomas was 100%; negative predictive value was 88% for cytologic and 82% for histologic material. Positive predictive value for metastatic carcinomas was 100%; negative predictive value was 100%. Seventy-two percent of the mesotheliomas revealed their greatest stemline within the range 1.80c-2.20c, whereas none of the metastatic carcinomas showed this stemline position. CONCLUSION: DNA image cytometry might be a very sensitive and highly specific, additional tool for identification of neoplastic cells in effusions as well as for the differential diagnosis of mesothelioma vs. metastatic carcinoma of the serous membranes.
Aneuploidy↗
Static DNA cytometry as a diagnostic aid in effusion cytology: II. DNA aneuploidy for identification of neoplastic cells in equivocal effusions.
OBJECTIVE: The sensitivity of conventional cytology for identification of neoplastic cells in effusions is unsatisfactory, about 58%. The rate of diagnostically equivocal effusions in routine cytology is about 6%. DNA aneuploidy has previously been proven to be a sensitive and specific marker for the identification of tumor cells in effusions. In the present study we determined if malignancy can be identified in cytologically equivocal cells in effusions using DNA aneuploidy as a marker, thus decreasing the rate of cytologically equivocal diagnoses in effusions. STUDY DESIGN: One hundred cytologically equivocal effusions of the serous cavities were obtained from routine diagnostic material. Nuclear DNA content was measured after Feulgen staining using a TV image analysis system. Data were correlated with patient follow-up. RESULTS: DNA aneuploidy was assumed if abnormal DNA stemlines, a coefficient of variation of the first DNA stemline > or = 10% or cells > 9c were observed. The sensitivity of DNA aneuploidy for the identification of malignancy was 55.9%. Specificity of DNA nonaneuploidy for benignity was 94.1%. The positive predictive value of the marker DNA aneuploidy for the occurrence of malignant cells was 97.9% since all but one DNA aneuploid case showed malignancy in follow-up. CONCLUSION: Image cytometry applying DNA aneuploidy as a parameter is able to detect the occurrence of malignant cells in cytologically equivocal effusions in about every second case. Thus, this method is able to increase diagnostic accuracy of conventional effusion cytology by decreasing the rate of diagnostically equivocal effusions.
Aneuploidy↗
Antioncogene P53 and mitogenic cytokine interleukin-8 aberrantly expressed in psoriatic skin are inversely regulated by the antipsoriatic drug tacrolimus (FK506).
Uncontrolled proliferation of epidermal cells is the most prominent characteristic of psoriasis. This widespread skin disease can be effectively treated with the microbial substance FK506, which acts by modulating gene expression. We, therefore, asked if the drug changes the expression of genes involved in growth regulation (the mitogenic cytokine interleukin-8 (IL-8) and p53, a negative cell cycle regulator) and signal transduction (protooncogenes c-ras, c-raf, and HER-2). Gene expression was monitored by semiquantitative mRNA-PCR and for p53 by immunocytochemistry in cultured primary keratinocytes (KC). In addition, p53 expression was analysed in skin biopsies of psoriatic patients. After 1-3 hr, IL-8 mRNA levels were dose-dependently decreased in tacrolimus (FK506)-treated cells. Protooncogene expression was not significantly altered. Interestingly, p53 transcription was clearly induced by FK506 treatment. This tendency could be verified on the protein level by immunocytochemistry. In contrast, p53 expression was decreased in lesional psoriatic as compared to normal skin, providing evidence that not only posttranslational modification of the p53 protein, but also transcriptional modulation of the p53 gene, are involved in pathological processes and pharmacological drug action in skin. Together with earlier results showing downmodulation for IL-8 receptor type A expression in cultured KC treated with FK506, these results suggest that both the mitogenic IL-8/IL-8R system and the cell cycle inhibitor p53 represent potential targets for the antipsoriatic action of the drug, whereas protooncogenes acting downstream in mitogenic signal transduction cascades are unaffected. The differential modulation of an entire set of genes provides evidence for the specificity of the drug effects and rules out nonspecific toxic effects on KC.
Dose-Response Relationship, Drug↗
PET-study of intracranial meningiomas: correlation with histopathology, cellularity and proliferation rate.
The glucose metabolism of 62 meningiomas was measured by fluorine -18-2-fluorodeoxyglucose (FDG) PET and correlated with proliferation rate (Ki-67 index) and tumor cellularity. The mean metabolic rate (MRGlu) for meningiomas was 0.26 +/- 0.13 mikromol/g/min (range 0.08-0.62 mikromol/g/min). The relative tumor FDG-uptake (Q-MRGlu) (tumor/contralateral cortex) of all meningiomas was calculated with 0.73 +/- 0.37 (0.24-1.79). Differences of Q-MRGlu were significant between the groups with high vs. low cellularity (p < 0.01), increased vs. normal proliferation rate (p < 0.025) and low (WHO grade I) vs. higher (WHO grades II, III) graded tumors. In recurrent meningiomas (14 tumors) the glucose metabolism was not increased. The data show that 18 FDG-PET is suitable to serve as non-invasive predictor of tumor growth characteristics in meningiomas.
Adult↗
Correlation of histology, cytogenetics and proliferation fraction (Ki-67 and PCNA) quantitated by image analysis in meningiomas.
Sixty meningiomas were classified histologically according to the WHO criteria and analyzed cytogenetically. The Ki-67 and PCNA proliferation fractions (PRFs) were assessed by immunhistochemistry. The staining results were quantified by TV-image analysis (Miamed, Leica, Germany). Within meningiomas of WHO-grade II/III and those with complete or partial deletion of # 1p, we found a significantly higher mean and maximal Ki-67 PRFs than in those of WHO-grade I and those with all other karyotypes. This was not the case for PCNA PRFs. No differences in PRFs were detectable between histological subtypes. Although these results were obtained after measurement of five high power fields (HPFs) only, the heterogeneity of PRF distribution within the tumors was high. Even after the measurement of 100 HPFs, the 95% confidence intervals were in a range of 18% to 34.3% PRF. This finding seems to be responsible for the moderate interobserver reproducibility of image analytical determination of PRF (r = 0.74). Nevertheless, there was a good correlation between subjective and objective image analytical assessment of PRF (r = 0.83). The significance of the maximum Ki-67 PRF per specimen implies the possibility of selecting those areas which show the highest fraction of positively stained nuclei for measurement, avoiding the problem of intraslide heterogeneity. Thus the time needed for image analytical quantification may be reduced.
Biomarkers, Tumor↗
DNA-aneuploidy as marker for neoplasia in G1-urothelial carcinomas.
Biopsies from 60 grade-1 urothelial carcinomas and 50 normal bladder mucosae have been investigated by DNA-image-cytometry after enzymatic cell separation to determine the diagnostic sensitivity of DNA-aneuploidy for the identification of low grade urothelial neoplasia. Two different modes to detect DNA-aneuploidy were compared: the conventional application of a threshold at 2.2c for the modal DNA value and the use of the Kolmogoroff-Smirnow (KS) test to compare GO/1 phase fractions of the cells under analysis and the internal reference cells. Whereas the specificity for both types of analysis was 100%, the sentitivity of the latter method was 73.3% compared to 48.3% of the conventional procedure. Therefore, the application of the KS-test for the identification of DNA- stemlines as aneuploid results in an increased detection rate was compared with the conventional threshold procedure. The high prevalence of DNA- stemline aneuploidy even in low grade urothelial cancers may qualify this marker as a possible aid in the cytologic evaluation of urine and bladder washings.
Aneuploidy↗
Mitotic frequency as a prognostic factor in breast cancer.
Grading of tumor malignancy in breast cancer should contribute essential information both for the prospective outcome of the individual patient as well as for TNM staging. In a series of 104 breast cancer patients we tested the prognostic validity and reproducibility of mitotic figure counting compared with TNM staging, Bloom and Richardson grading, DNA single cell cytometry, and morphometry. Four-micrometer thick hematoxylin-eosin-stained routine slides were investigated. Mitotic figures were counted in representative areas of the tumor in 10 159-microns 2-sized high power fields (HPFs) at a 400X magnification; the median value was seven and the threshold for the 25th percentile was three. This value should replace the common but prognostically invalid threshold of 10. Univariate survival analysis showed that mitotic figure counting allows the identification of three groups of patients (< or = 3, 4 to 20, > 20 mitoses per HPF) with significantly different survival probabilities (P < .0001; P = .0178). Depending on the number of mitotic figures, length of survival was significantly different within the group of T1N0 tumors (P = .0082) and the group of T1N1 or T2N0 tumors (P = .0251). In a Cox stepwise regression model mitotic frequency counting added prognostic information to tumor size and was of higher prognostic significance than lymph node status, DNA ploidy, or mean nuclear area. The 95% confidence limit for interobserver reproducibility, tested in 20 cases, was plus/minus 8 mitoses. After quartilization an agreement of 75% was observed.
Aged↗
Aneuploidy in actinic keratosis and Bowen's disease--increased risk for invasive squamous cell carcinoma?
The value of DNA single cell cytometry for the detection of aneuploidy was assessed in 100 specimens of actinic keratoses and 39 specimens of Bowen's disease. Ten seborrhoeic keratoses and 10 samples of normal epidermis served as negative control groups. Monolayer smears, prepared from formalin-fixed, paraffin-embedded tissues, were Feulgen-stained and used for interactive DNA-cytometry. In each specimen, the DNA content of 150 randomly chosen squamous epithelial cells was measured, using a TV-image analysis system (TAS-plus, Leica, Germany). Aneuploidy was diagnosed if at least three nuclei with a DNA content above 5c (5cEE > or = 3) were found. The aneuploidy rate in actinic keratosis was 69% (69 of 100) and in Bowen's disease was 95% (37 of 39). Another 20 specimens of actinic keratoses and the remaining two specimens of Bowen's disease were diagnosed as suspicious for aneuploidy (0 < 5cEE < 3). The 20 specimens of seborrhoeic keratoses and normal epidermis did not show any nuclei above the 5c level, and were classified as non-aneuploid. This indicates a sensitivity of 76% (106 of 139) and a specificity of 100% (20 of 20). The frequent occurrence of aneuploidy in actinic keratoses and Bowen's disease underlines the character of the lesions as epidermal carcinomas in situ, but does not explain the long-term low incidence of invasive growth.
Aneuploidy↗
DNA aneuploidy, increased proliferation and nuclear area of plasma cells in monoclonal gammopathy of undetermined significance and multiple myeloma.
DNA cytometric and morphometric parameters of plasma cells were determined in 73 multiple myelomas (MMs), 31 monoclonal gammopathies of undetermined significance (MGUS), 11 reactive plasmacytoses (RPs) and 33 cases of normal bone marrow (NBM) using a TV-based static cytometer combined with a computerized relocation stage. Neoplastic transformation was defined by either an aneuploid DNA profile, an increased proliferative fraction or an increased mean nuclear area of plasma cells. Using threshold values defined by mean values and variations of NBM and RP, 67/73 MMs were correctly classified as malignant (sensitivity, 92%), whereas all NBMs and RPs were classified as benign (specificity, 100%). In MGUS, cytometric parameters of malignancy were detected in 19/31 cases (61%). Six of these cases developed MM after a median time of 4.9 years (mean follow-up, 9.3 years). Another three cases with MGUS developed MMs without previous cytometric plasma cell aberrations. Cytometric data from repeated measurements in MGUS progressing to MM exhibited perfect consistency over time concerning the presence or absence of cytometrically detected neoplastic transformation. Cytometric aberrations seem to be frequently associated with neoplastic growth in monoclonal plasma cell proliferations but do not predict clinically malignant behavior.
Adult↗
Consensus report of the ESACP task force on standardization of diagnostic DNA image cytometry. European Society for Analytical Cellular Pathology.
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DNA, Neoplasm↗
Consensus report of the European Society for Analytical Cellular Pathology task force on standardization of diagnostic DNA image cytometry.
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Cell Nucleus↗
Polyploidy in non-neoplastic tissues.
AIM: To investigate the prevalence and amount of polyploidy in fine needle aspiration specimens of the liver, urinary cytospin preparations, and cytospin preparations from pleural and peritoneal fluid. METHODS: Cells from 44 liver smears, 48 urine specimens, and 46 pleural and peritoneal aspirations were examined. After Feulgen restaining the DNA content of 100 randomly selected nuclei was determined using a TAS-plus image analysis system, combined with an automated microscope. RESULTS: Polyploidy was observed up to 16c in the liver, and up to 8c in urothelium and mesothelium. Sixty eight per cent of the liver aspirates contained polyploid nuclei. The rate in urothelium was 20.8% and in mesothelium 6.5%. CONCLUSIONS: Polyploidy in the liver may be interpreted as being associated with tissue differentiation, but the findings in urothelium and mesothelium remain of unknown importance.
Aged↗
DNA aneuploidy in G1-urothelial carcinomas of the urinary bladder.
Thirty G1-urothelial carcinomas of the bladder were investigated using DNA cytophotometry. Specimens obtained by tumour resection and quadrantic biopsy were first fixed in formalin and then embedded in paraffin. The cells were then separated using the method developed by Hedley. The DNA content was established after Feulgen staining using a TV-based image analysis system. DNA aneuploidy, a marker for neoplasia, was investigated using the so-called stemline interpretation developed by Böcking. This method compares the DNA content of the G0/1 tumour cells in the cell population to be analysed with the reference cell population, using the Kolmogoroff-Smirnow test (p < 0.001). On this basis, 23 tumours (76.6%) were DNA aneuploid, whereas if DNA aneuploidy is defined (as conventionally) as a DNA content of > 2.2c, only 17 tumours (56.6%) were found to be DNA aneuploid. The mean stemline value was 2.4c (range 1.85-3.96). By using a more precise TV-based image analysis system and this new DNA stemline interpretation, the rate of detection of DNA aneuploidy was improved by 20%. The diagnosis of G1-urothelial carcinomas by DNA cytophotometry is at least 25% more sensitive than that using conventional cytology (20-50%). We suggest that modified DNA cytophotometry could be a useful, additional diagnostic tool in examining doubtful urinary samples.
Adult↗
Clinical utility of cellular DNA measurements in prostate carcinoma. Consensus Conference on Diagnosis and Prognostic Parameters in Localized Prostate Cancer. Stockholm, Sweden, May 12-13, 1993.
At a WHO consensus conference on Early Diagnosis and Prognostic Parameters in Localized Prostate Cancer, a working group discussed the clinical utility of DNA measurements in stages T2 and T3 prostate carcinoma. Incidentally discovered prostate cancer of stage T1 was excluded. The members of the working group, representing various clinical and laboratory disciplines, discussed technical considerations of DNA measurements by flow and image analysis, pretreatment prediction of prognosis, and posttreatment clinical relevance. The group agreed to subdivide tumors into diploid, tetraploid and non-tetraploid aneuploid, expressing various degrees of aggressiveness and gave guidance for the definition of limits of these groups. The panel agreed that knowledge on DNA ploidy prior to treatment is of value in treatment decisions, particularly when surveillance is a treatment option. Aneuploid tumors can be expected to respond very poorly to either irradiation or endocrine therapy, and the presence of aneuploid tumor, either on pretreatment biopsies or in radical prostatectomy specimens, is an ominous sign. The identification of a group of patients with a uniformly poor prognosis should encourage medical oncologists and basic scientists to develop adequate treatment options for this particular group. The panel expressed a strong opinion that DNA ploidy should be uniformly studied in clinical trials, particularly in patients with localized prostate cancer.
Biopsy↗
Quantitative analysis of the neu oncogene in normal and transformed epithelial breast cells by fluorescence in situ hybridization and laser scanning microscopy.
In this study we evaluated indirect fluorescence in situ hybridization of the neu oncogene in combination with laser scanning microscopy for the detection and quantification of fluorescence signals in single cells. Cell lines from human tumor tissue with neu oncogene amplification and one nonneoplastic human epithelial cell line from a lactating breast with a neu single copy as the reference were used. After quantitative fluorescence analysis of three mixed cell populations (neoplastic cells and reference cells), we evaluated the cell morphology and location of the fluorescence signal by laser scanning microscopy. Finally, the number of oncogene copies in the tumor cell population could be estimated from the ratio of its mean background-corrected integrated fluorescence intensities to those of the reference cells. In all mixed cultures the ratio of fluorescence intensities was within the limits of oncogene copy numbers known from the literature and evaluated by southern blot analysis. The lowest oncogene copy number possible to quantify was four to eight genomic copies.
Breast Neoplasms↗