DNA grading of malignancy in breast cancer. Prognostic validity, reproducibility and comparison with other classifications

The prognostic significance of the "DNA malignancy grade" (DNA-MG) was tested in a series of 104 breast cancer patients in comparison with TNM staging, histomorphologic grading according to Bloom and Richardson, mean nuclear area (MNA) and DNA-histogram classification according to Auer. The reproducibility and representativity of the grading systems were investigated, and their results in primary tumors and lymph node metastases were compared. The scalar DNA-MG was assessed on monolayer smears prepared from paraffin-embedded tissues; the smears were automatically Feulgen stained and used for rapid interactive DNA cytometric evaluation by an automated microscope and a TV image-analysis system. TNM staging showed the highest correlation with survival, followed by histomorphologic grading and DNA-MG; MNA and the DNA-histogram classification failed to give statistically significant prognostic information. Both histomorphologic grading and DNA-MG were identified as parameters adding independent prognostic information to the TNM staging. However, only DNA-MG demonstrated an acceptable reliability, with small 95% ranges between repeated measurements within the primary tumor (+/- 0.3 DNA-MG) and a strong correlation between the results in the primary tumor and its lymph node metastases. These findings show that the DNA-MG is a valid and reliable prognostic index that adds significant prognostic information to TNM staging.     

Prognostic biomarkers in renal cell carcinoma: relevance of DNA ploidy in predicting disease-related survival

OBJECTIVE: To investigate the prognostic value of DNA ploidy, Ki-67 index and p53 expression in relation to disease-related survival in a consecutive series of patients with renal cell carcinoma (RCC). MATERIAL AND METHODS: The study group consisted of 64 RCC patients treated by radical nephrectomy. Histological type, pathological staging and nuclear anaplasia were assessed according to the WHO classification, TNM system and Fuhrman grading criteria, respectively. Ploidy was determined by DNA flow cytometry using two sampling methods (frozen vs paraffin-embedded tissue). Ki-67 and p53 were evaluated by immunohistochemistry techniques using two cutoff points (10% vs mean value) for staining interpretation. Kaplan-Meier and Cox regression analyses were used for prognostic evaluation. RESULTS: Thirty-one tumors (48.4%) showed DNA diploidy and 33 (51.6%) were DNA aneuploid. Concordance between both ploidy measurement methods was found in 85.5% of cases (p=0.0455). The mean values for Ki-67 and p53 immunostaining were 3.65% (0-23.5%) and 5.90% (0-55.9%), respectively. DNA ploidy significantly correlated with staging, tumor size (pT), nuclear grading, and Ki-67 (mean value cutoff). Ki-67 (10% cutoff) correlated with staging and pT, while p53 (mean value cutoff) was associated with Ki-67 (mean value cutoff). There were significant differences between survival curves for pathological stage, pT, nuclear grade, ploidy, Ki-67 (both cutoffs), and p53 (10% cutoff). By univariate regression analysis, stage III and stage IV, pT3, aneuploidy, high Ki-67 (both cutoffs), and p53 overexpression (10% cutoff) showed significant correlations with worse disease-related survival. In addition, DNA aneuploidy significantly correlated with poor prognosis within stages I/II (p=0.0355) and stages III/IV (p=0.0138) of the disease. CONCLUSION: The results indicate that DNA ploidy has relevant prognostic value in RCC, adding useful information to the classic histopathological indicators of clinical outcome.     

Clinical applications of image cytometry to human tumour analysis

Image cytometry (ICM) is widely applied to the automated screening, the detection, the diagnosis, the classification, the prognosis and the therapeutic follow-up of different types of cancers (breast, bladder, cervix,...). This review describes the analysis methods and the applications of nuclear image analysis, the determination of DNA content and the analysis of morphometry and of nuclear texture. DNA content analysis can contribute to a prognostic information in addition to other prognostic factors for breast, renal and prostate cancers. For ovarian cancer, aneuploidy seems to be related to prognosis. Bladder tumours with DNA aneuploidy were frequently of high malignancy while ploidy was significantly correlated to relapse risk. For digestive cancers, patients presenting DNA diploid tumours show a better survival than patients with aneuploid ones. Morphometry seems to be a more important criterion than other conventional prognostic factors of invasive breast and digestive carcinomas. A differential diagnosis between normal and neoplastic thyroids is more precise when based on a quantitative evaluation of texture associated to morphometry. Textural parameters permit the discrimination of two populations of patients having a different prognosis and could thus be an aid for prognosis in prostatic cancers. Morphonuclear parameters contribute to separate low and high grade bladder carcinomas. Although ICM was frequently reported, results from the reported examples were not always obvious. In conclusion, the measurements obtained with ICM could be helpful for a decision in several cancers but could not be a substitute for the classical approach of the pathologist.     

Value of morphometric nuclear image analysis using the Feulgen reaction in renal cell carcinoma

OBJECTIVE: To evaluate retrospectively the ability of morphometric nuclear image analysis to predict survival in patients with renal cell carcinoma. STUDY DESIGN: The subjects were 40 patients with previously untreated renal cell carcinoma. Pathologic stage was determined using Robson's stage system. Nuclear grade was assigned according to the criteria of Fuhrman et al. We used the Feulgen staining technique, which has been widely used for the histochemical assessment of nuclear DNA content. A minimum of 300 nuclei were analyzed from each subject. Five variables in morphometric nuclear image analysis were measured: nuclear area, nuclear perimeter, nuclear ellipticity, nuclear regularity and DNA content. Cox's proportional hazard model was applied to identify prognostic usefulness with respect to survival time. RESULTS: All nuclear morphometric variables but nuclear regularity correlated with tumor grade. According to univariate survival analyses, Robson stage and nuclear ellipticity revealed a prognosis on survival with statistical significance. After adjustments for age and sex, nuclear ellipticity remained the only significant prognostic factor related to survival (P < .01). The survival rates were relatively high for patients with nuclear ellipticity > 773 as compared to those with nuclear ellipticity < 773 (P < .05). CONCLUSION: These findings indicate that morphometric nuclear image analysis using the Feulgen reaction is a reliable and efficient technique and that nuclear ellipticity is the most discriminating morphometric variable for predicting the prognosis of renal cell carcinoma patients.     

Prognostic implication of histological oligodendroglial tumor component: clinicopathological analysis of 111 cases of malignant gliomas

The favorable prognosis of high-grade oligodendroglial tumor such as glioblastoma (GBM) with oligodendroglioma component (GBMO) has been suggested; however, the studies which examine the prognostic significance of oligodendroglial tumor were limited. In this study, we performed a histopathology-based reevaluation of 111 cases of high grade gliomas according to the latest World Health Organization (WHO), and compared the clinical outcomes between oligodendroglial tumors and pure astrocytic tumors. The survival analysis revealed that the patients with high grade oligodendroglial tumor including GBMO significantly indicated better prognosis compared to the patients with high grade pure astrocytic tumors (GBM and AA, anaplastic astrocytoma) as expected, and the obtained survival curves were almost identical to those from the patients with conventional Grade III or Grade IV tumors, respectively. Moreover, if the cases of oligodendroglial tumor were histopathologically excluded, the patients with AA exhibited extremely poor prognosis which was similar to that of GBM, suggesting that the histological identification of oligodendroglial tumor component, even partially, prescribe the prognosis of high grade glioma patients. This is the prominent report of retrospective clinicopathological analysis for high-grade gliomas throughout Grade III and IV, especially referring to the prognostic value of histological oligodendroglial tumor component; in addition, our results might offer an alternative aspect for the grading of high-grade astrocytic/oligodendroglial tumors.     

Preparation of cells from paraffin-embedded tissue for cytometry and cytomorphologic evaluation

A method is described for the preparation of monolayer smears from paraffin-embedded tissue. The smears are suitable for automated image analysis and DNA measurements while still allowing interpretation of nuclear morphology. The proposed technique uses enzyme treatment and syringing for cell dispersal. The preparation of cell monolayers is performed by cytocentrifugation. After staining the specimens with gallocyanin, nuclear DNA can be measured. Automated DNA measurements using the Leyden Television Analysis System (LEYTAS) showed coefficients of variation of 4.5% for the diploid cell population of suspended benign tissue. After DNA measurements, the specimens are counterstained using orange G and eosin. Since gallocyanin has spectral properties similar to those of hematoxylin, the obtained end product is comparable to specimens stained according to the routinely used Papanicolaou procedure. Using this technique, image cytometry can be applied to paraffin-embedded tissue in combination with conventional cytomorphologic study of the cells.     

Clinical aspects for the use of DNA image cytometry in detection of bladder cancer: a valuable tool?

The employment of DNA flow or image cytometry for oncological diagnostic procedures is favored because of its high correlation to tumor biological behavior. Prognostic statements and therapeutic strategies therefore are based on the high validity of DNA cytometric measurements. Using 151 bladder washings from patients suspected of bladder cancer for this study, we examined the clinical value of various common methods of DNA single cell (SCI) and stemline interpretations (SLI). Comparing the specificity and sensitivity of DNA image cytometry in detection of bladder tumors, we found 81 and 52%, respectively, for SCI of Boecking, 84 and 45% for SLI of Boecking, 61 and 58% for SLI of Fu, and 82 and 40% for conventional stemline interpretation. To improve diagnostic and prognostic validity of DNA image cytometry, we designed our own method of interpretation. In consequence, we identified six single DNA parameters out of all recorded measurements that correlated most to histopathological grading (G1-G3). Creating reference values at random and rating by points, we used a cytometric grading system for ranking. In detection of bladder cancer specificity and sensitivity ultimately arrived at almost 70% in application of our method. Thus, by this study, we were able to show that sensitivity of DNA examination can be increased by combining various DNA parameters. Apart from our own scheme, the discrepancy in interpretation of DNA image cytometry does not allow us to recommend this procedure as the only diagnostic in detection of bladder cancer. However, in regard to prognostic statements, particularly tumor biological behavior, DNA image cytometry appears to be useful.     

Pathologic significance of proliferative activity and oncoprotein expression in astrocytic tumors

OBJECTIVE: To clarify the clinicopathologic significance of immunohistochemistry for proliferative activity and oncoprotein expression in astrocytic tumors. STUDY DESIGN: Ninety-seven cases of brain astrocytic tumors with histologic grading and follow-up data were investigated with immunohistochemistry and image analyzer to detect the expression of proliferating cell nuclear antigen (PCNA), silver-binding nucleolar organizer regions (AgNORs) and several oncoproteins. RESULTS: PCNA was significantly related to AgNORs, grading and prognosis of astrocytomas. The frequency of mutant p53 protein expression was higher in grade 2-4 astrocytomas than in grade 1. Epidermal growth factor (EGF) (37.1%), EGF receptor (83.5%) and p21ras (42.3%) expression levels were related to neither the grade nor prognosis of the tumors. The positive ratios of p53 antibodies were higher in grades 2-4 than in grade 1, and the intensities correlated with PCNA but not with prognosis. CONCLUSION: Aberrations of c-erbB-2, p21ras, EGF and EGF receptor might be early events in the initiation and progression of astrocytomas, whereas p53 overexpression is involved in all the stages. Immunohistochemical detection had no prognostic value. PCNA could be important to the evaluation of astrocytoma malignancy.     

Standardized AgNOR analysis as a prognostic parameter in endometrial carcinoma, endometrioid type

OBJECTIVE: To evaluate interobserver reproducibility of histologic grade in endometrial adenocarcinomas of endometrioid type (EC), to assess the relationships between nuclear grade and the amount of argyrophilic nucleolar organizer region (AgNOR) proteins and to determine the prognostic value of AgNOR proteins and the main clinicopathologic parameters. STUDY DESIGN: Architectural and nuclear grading were independently assessed by two pathologists in 64 formalin-fixed, paraffin-embedded surgical samples of EC obtained from an equal number of patients (age range, 38-84 years; mean, 63.5). Interobserver agreement was determined using the kappa statistic; discrepant cases were reviewed, and a consensus was reached. Standardized AgNOR analysis was performed according to the guidelines of the Committee on AgNOR Quantification, measuring the mean area of AgNORs per nucleus (NORA) by an image analysis system. RESULTS: The kappa values for interobserver agreement were substantial for architectural grading and moderate for nuclear grading. When NORA values were compared to the nuclear grade assessed by different observers, the most significant linear correlation (r = .713, P < .001) was found for the nuclear assessment obtained by consensus of the two pathologists. Moreover, statistical analysis allowed discrimination of architectural grade 1 from grade 2 and 3 EC. By the Kaplan-Meier method, the prognosis was worse for patients with higher NORA values (> 4.212 micron 2), while, by Cox multivariate analysis, AgNOR quantity emerged as an independent prognostic variable. CONCLUSION: Use of standardized AgNOR analysis may be an additional and objective tool in the assessment of histologic grade as well as a reliable method of determining prognosis in EC.     

Cytological grading of breast cancer in Giemsa-stained fine needle aspiration smears

A potential cytological nuclear grading based on a semi-quantitative evaluation of three basic nuclear features, size of cell nuclei, anisonucleosis and the proportion of nucleoli-containing-nuclei, was tested on 74 Giemsa-stained fine needle aspiration of breast smears for its reliability in establishing the malignant potential of breast cancer. The prognostic impact of DNA-ploidy and S-phase fraction was also assessed. A good correlation between the three basic nuclear features, DNA-ploidy, S-phase fraction, cytological nuclear grade and histological grade, was shown. Using the cytological nuclear grade proposed, correct classification of cases between low histological grade (HG I) and high histological grade (HG II + HG III) was achieved in 79.73%. A statistically significant difference in 5-year survival rate was also observed between low malignancy grade and high malignancy grade breast cancer patients, regardless of the grading method used. DNA-ploidy and S-phase fraction were not statistically significant in establishing the malignant potential of breast cancer.     

Cytologic grading and DNA image cytometry of breast carcinoma on fine needle aspiration cytology smears

OBJECTIVE: To correlate the cytologic grade of breast carcinoma with DNA image cytometry (ICM) and nuclear area on fine needle aspiration cytology (FNAC) smears. STUDY DESIGN: In this prospective study, FNAC material from 28 breast carcinomas were studied for cytologic grade and DNA ICM. Breast carcinomas were classified as grade 1-3 (low to high). DNA histograms were classified by the modified Auer method. Degree of hyperploidy (DH), ploidy balance (PB) and nuclear area (NA) were measured on Feulgen-stained smears by a CAS 200 image cytometer. Cytologic grade was correlated with DNA ICM findings and NA. RESULTS: There were 3 cytologic grade 1, 13 grade 2 and 12 grade 3 breast carcinomas. Seven of eight cases of hypertetraploid aneuploidy were grade 3 tumors. All cytologic grade 1 tumors were diploid. There were significant differences in DH, PB and NA in different grades of breast carcinoma (one-way ANOVA). CONCLUSION: DNA image cytometry in combination with cytologic grading might offer additional information for the characterization of breast carcinomas diagnosed by FNAC. These observations are of particular interest with the introduction of preoperative chemotherapy.     

Malignancy index based on flow cytometry and histology for renal cell carcinomas and its correlation to prognosis

Fifty-five human renal cell carcinomas, removed by nephrectomy, were classified using flow cytometry and histology. The parameters obtained by flow cytometry were DNA index, fractions of cells in the phases, and fractions of tumor cells versus normal cells. From histology, the routine classification of tumor structure and morphology were obtained, and a nuclear grading according to Arner et al. (Acta Chir Scand [Suppl] 346:1-12, 1965) was determined. All parameters including tumor stage according to Robson et al. (J Urol 101:297-301, 1969) were subjected to discriminant analysis in order to define a malignancy index. The patients were divided into two groups with good and poor prognosis (low and high risk). Those patients who were free of multiple metastases in 2 years and more after nephrectomy were assigned to the group "good prognosis"; those who developed multiple metastases for 2 years represented the "poor prognosis" group. The malignancy index determined by discriminant analysis yielded 91% correct predictions of prognosis.     

Prognostic significance of DNA image cytophotometry for osteosarcoma

OBJECTIVE: To investigate the prognostic significance of DNA image cytophotometric data. STUDY DESIGN: Twenty-six osteosarcomas in patients without lung metastases were investigated for several cytophotometric data. In 24 cases, these data were correlated with the clinical course of the patients to assess the prognostic value of nuclear DNA content in osteosarcomas. RESULTS: Of all osteosarcomas, 96% showed aneuploid DNA content. Patients with tumors having a 2c deviation index (2cDI) of 12.00, DNA malignancy grade (DNA-MG) of 2.0, a mean DNA content (MDC) of 4.95 c, DNA index (DI) of 1.75 or mean nuclear area (MNA) of 130 microns 2 had a significantly lower overall survival rate as compared to those with lower values (P < .05). CONCLUSION: Image cytophotometric features, such as 2cDI, DNA-MG, MDC, DI and MNA, are of prognostic value in patients with osteosarcoma and free of lung metastases.     

Neural network-based segmentation and classification system for automated grading of histologic sections of bladder carcinoma

OBJECTIVE: To develop an image analysis system for automated nuclear segmentation and classification of histologic bladder sections employing quantitative nuclear features. STUDY DESIGN: Ninety-two cases were classified into three classes by experienced pathologists according to the WHO grading system: 18 cases as grade 1, 45 as grade 2, and 29 as grade 3. Nuclear segmentation was performed by means of an artificial neural network (ANN)-based pixel classification algorithm, and each case was represented by 36 nuclei features. Automated grading of bladder tumor histologic sections was performed by an ANN classifier implemented in a two-stage hierarchic tree. RESULTS: On average, 95% of the nuclei were correctly detected. At the first stage of the hierarchic tree, classifier performance in discriminating between cases of grade 1 and 2 and cases of grade 3 was 89%. At the second stage, 79% of grade 1 cases were correctly distinguished from grade 2 cases. CONCLUSION: The proposed image analysis system provides the means to reduce subjectivity in grading bladder tumors and may contribute to more accurate diagnosis and prognosis since it relies on nuclear features, the value of which has been confirmed.     

DNA quantification in colorectal carcinoma using flow and image analysis cytometry

Numerous studies using flow cytometry (FCM) have shown that DNA quantification and ploidy classification can provide information of prognostic significance for patients with colorectal carcinoma; recent advances in image analysis cytometry (image cytometry, ICM) provide a new, alternative technique for DNA quantification. This study investigated whether (1) patients with colorectal carcinomas that exhibit a diploid pattern of DNA distribution have improved five-year survival statistics as compared to their non-diploid counterparts and (2) ICM provides quantitative data comparable to that obtained by FCM. DNA quantification and ploidy classification of 27 cases of primary colorectal carcinoma was performed on archival paraffin-embedded tissue by both FCM and ICM; 70% (19) of the tumors were classified as nondiploid by ICM while 56% (15) were similarly classified by FCM. Diploid tumors were associated with Dukes' stage A while nondiploid tumors were associated with Dukes' stage D. The overall five-year survival rate was 75% for patients with ICM diploid tumors and 67% for patients with FCM diploid tumors. The five-year survival was only 53% for patients with nondiploid tumors identified by both techniques. This study confirmed that DNA quantification is an important prognostic indicator for patients with colorectal carcinoma. It also showed that ICM provides data comparable to that of FCM and may be more sensitive.     

Mean nuclear area of fine needle aspirates of primary preoperative palpable breast carcinoma using image cytometry

OBJECTIVE: Early, operable breast cancers in appropriate patients are increasingly being treated preoperatively using neoadjuvant chemotherapy. A good response rate is seen with high grade tumors. Nuclear size, which may reflect the grade of the tumor, is also of possible prognostic value in breast cancer. STUDY DESIGN: We measured the mean nuclear area (MNA) of 114 consecutive preoperative fine needle aspirates of palpable, operable breast cancers. We used computerized image cytometry to measure nuclear area to determine tumor biology and possible grade prior to treatment. RESULTS: Histologic grade distribution was as follows: low grade, 15%; moderate grade, 40%; and high grade, 45%. Mann-Whitney test for trend on tumor size and histologic grade between MNA showed a significant relationship between MNA and tumor size (P=.016) but no significance between MNA and histologic grade (P =.22). The chi2 and Fisher Exact Test between MNA and node-positive or -negative patients showed no significance. CONCLUSION: When correlating MNA with tumor size and histologic grade, high MNA is present at a higher frequency as tumor size and histologic grade increase.     

Prognostic significance of DNA cytometry in carcinoma of the uterine cervix FIGO stage IB and II.

OBJECTIVE: To assess the prognostic value of DNA-image cytometry in cervical carcinoma of the uterus and its relation to other established prognostic factors. STUDY DESIGN: The study included 116 cases of cervical carcinoma FIGO stages IB and II which were treated with radical abdominal hysterectomy. The median follow-up was 55 months (range 1-162 months). DNA image cytometry was performed on cytologic specimens prepared by enzymatic cell separation from formalin-fixed, paraffin-embedded tissues. DNA stemline ploidy, DNA stemline aneuploidy, 5c exceeding rate, 9c exceeding rate, 2c deviation index, and DNA malignancy grade were computed. DNA-variables as well as various clinical and histological variables were related to survival rates. RESULTS: In multivariate statistical analysis DNA stemline ploidy using 2.2c as a cut-off value and FIGO stage showed to be statistically significant available presurgery predictors of survival, whereas the postsurgical parameters lymphonodal status, tumor size and parametrial involvement were significantly correlated with survival. The synopsis of all parameters in a multivariate Cox model indicated that - with declining relevance - the number of positive pelvic lymph nodes, DNA stemline ploidy using a cut-off level at a modal value of 2.2c, largest pelvic lymph node, 5c exceeding rate, and ratio of carcinoma area to cervix area, were of predictive value for survival. CONCLUSIONS: Our results suggest that prognostic information deducted from classical staging parameters is successfully complemented by DNA image cytometry which can be applied pretherapeutically.     

Prognostic significance of image cytometry DNA parameters in tissue sections from breast and gastric cancers.

The ploidy patterns determined for several groups of mammary and gastric carcinomas were subjected to a set of statistical analyses. The DNA distribution patterns were derived from image cytometry measurements of each of at least 150 Feulgen-stained tumour cell nuclei from tissue sections from 84 invasive ductal carcinomas of the breast and from 30 tubular adenocarcinomas of the stomach. Widely used DNA parameters (mean value, standard error of the mean, DNA-malignancy grade, 2c deviation index and the exceeding rate according to B?cking, DNA-histogram types according to Auer, DNA-index according to Atkin) were analysed by univariate and multivariate statistics. The DNA histograms were also analysed multiparametrically. The results showed different prognostic groups of the breast tumours to be distinguishable on single parameters with a reliability of up to 66%. None of these parameters permitted the discrimination of gastric carcinomas. Although the DNA-histogram-analysis increased accuracy by nearly 10%, compared with the classification accuracy of the best single parameters, it is still far from being applicable in clinical diagnostics. The use of further image cytometry parameters will be required for such applications.     

DNA ploidy and cell cycle analysis in ear malignant melanoma by flow and image cytometry.

Sixteen ear malignant melanomas (MM) were studied for ploidy and cell cycle analysis by flow and image cytometry. The results were compared with clinical (age, sex, stage), histologic (depth of invasion, level, type) and prognostic (recurrence, death) parameters. Single nuclear suspensions were obtained from fixed, paraffin-embedded tumor and adjacent normal tissue processed separately according to Hedley's technique. These, a "spiked" specimen of normal tissue and tumor, and a spleen diploid control were analyzed on a FACScan flow cytometer (Becton Dickinson, Mountain View, California, U.S.A.). Feulgen-stained Cytocentrifuge preparations of nuclear suspensions of normal, MM and diploid spleen were analyzed with the CAS 200 Image Analyzer (Cell Analysis Systems, Inc., Elmhurst, Illinois, U.S.A.) against commercial calibration rat hepatocytes defined as diploid. Six (37.5%) MM were diploid, and 10 (62.5%) were aneuploid; 8 (90%) were hypodiploid, for a high frequency. There were no statistically significant correlations between clinical, pathologic, prognostic or cell cycle analysis parameters and ploidy, although poor prognostic features tended to be in aneuploid lesions.     

Image cytometry of aneuploidy, growth fraction (MoAb Ki-67) and hormone receptors (ER, PR) immunocytochemical assays in breast carcinomas

DNA nuclear content was assessed in human breast carcinomas (n = 132) using image cytometry. Optical density histograms of Feulgen stained cell imprints from fresh tissue samples, subsequently frozen for immunocytochemical assays, were determined by the SAMBA system and used for the DNA index, the ploidy balance (PB) and the proliferation index (PI) computation. The three parameters were correlated to (i) histological data (tumour grade, vascular and/or lymph node invasion) and to (ii) growth fraction (Ki67), hormone receptor antigenic sites (ER, PR) and intramedullar (bone marrow) biopsies and anti-KL1-positive epithelial cells. It was shown that 57% of breast carcinomas were aneuploid. Aneuploidy PI significantly correlated to the criteria of poor prognosis such as high tumour grade, vascular and lymphatic invasion and to increased Ki67-positive cells, and the absence of or low ER and PR. Since image cytometry is easy to handle and perfectly suitable for current diagnostic practice in pathology departments, particularly for tumour cell ploidy assessment and standardized analysis of immunostaining procedures with morphological control of the preparation, we conclude that image cytometry, as performed with the SAMBA, must be regarded as a relevant tool for prognosis evaluation and therapy guidance in individual patients.