Intraepithelial CD8-positive T lymphocytes predict survival for patients with serous stage III ovarian carcinomas: relevance of clonal selection of T lymphocytes.

BACKGROUND: The aim of this study was to investigate the prognostic effect of tumour-infiltrating lymphocytes (TILs) in serous stage III ovarian carcinoma to determine TIL clonality and to correlate this to Her2/neu expression. METHODS: Formalin-fixed and paraffin-embedded ovarian carcinomas were examined for CD20-, CD3-, CD4- and CD8-positive lymphocytes (n=100), and for Her2/neu-positive tumour cells (n=55/100) by immunohistochemistry. Clonality analysis was carried out by T-cell receptor gamma (TCRgamma) gene rearrangements (n=93/100). Statistical analyses included experimental and clinico-pathological variables, as well as disease-free (DFS) and overall (OS) survival. RESULTS: CD20-positive B lymphocytes were present in 57.7% (stromal)/33.0% (intraepithelial) and CD3-positive T lymphocytes in 99.0% (stromal)/90.2% (intraepithelial) of ovarian carcinomas. Intraepithelial CD3-positive T lymphocytes were correlated with improved DFS in optimally debulked patients (P=0.0402). Intraepithelial CD8-positive T lymphocytes were correlated with improved OS in all optimally debulked patients (P=0.0201) and in those undergoing paclitaxel/carboplatin therapy (P=0.0092). Finally, rarified and clonal TCRgamma gene rearrangements were detected in 37 out of 93 (39.8%) and 15 out of 93 (16.1%) cases, respectively. This was marginally associated with improved DFS (P=0.0873). Despite a significant correlation of HER2/neu status and intraepithelial CD8-positive lymphocytes (P=0.0264), this was non-directional (R=-0.257; P=0.0626). CONCLUSION: Improved survival of ovarian cancer patients is related to the infiltration, clonal selection and intraepithelial persistence of T lymphocytes.

Stereological characteristics of the equine accessory nerve.

Stereological techniques have been increasingly employed for assessment and characterization of neuromuscular diseases in humans and animals. As an adjunct to histopathology, morphometrical algorithms provide quantitative evidence of the peripheral nerve composition, thereby shedding light on its fibre characteristics and basic electrophysiological properties. In the horse, stereological investigations already have focussed on the recurrent laryngeal, deep peroneal and lateral palmar nerves (LPN). Of these, only the latter is suitable for taking biopsies in clinical settings, however, it does not contain any motor fibres and Ia-afferents. On account of its virtually mixed fibre qualities, most researchers today recommend the cervical branch of the equine accessory nerve (AN) for harvesting diagnostic samples. Thus, the present study was carried out to gain morphometrical proof of the AN composition and to obtain stereological base values in healthy individuals using state-of-the-art technology. All parameters were compared to the common peroneal nerve (CPN), known to harbour all myelinated fibre classes. As this second biopsy site is located farther distally to the neuro-axis, attention was paid to possible length-dependent features. Taken together, digital image analysis could be accurately applied on all AN samples. Stereology supported the histological and clinical evidence that the AN contains all myelinated fibre types. The huge range and scatter of fibre counts and density (3351-17,812/mm(2)) per fascicle were comparable to that measured in the equine common peroneal, deep peroneal, lateral palmar and recurrent laryngeal nerves. Similar to those, fibre diameter distribution was bimodal with slow Abeta- and Agamma-mechanoceptor afferents outnumbering large myelinated Aalpha-fibres by a factor of about 1.5. With a g-ratio at 0.55 +/- 0.001, the overall degree of myelination in the AN is highly consistent and insignificantly ranges between that of the equine common peroneal and LPNs. Apart from this subtle deviation, a statistically relevant difference between the more proximal AN and the distal CPN could not be documented. By obtaining morphometrical standard parameters and even more sophisticated distribution indices, stereology is a valuable tool for detection of subtle changes that are likely to escape from the investigators' eyes. The AN serves as a reliable source for advanced peripheral nerve research and should be accompanied by farther distal nerve probes for assessment of neuropathies that present with a proximodistal gradient.

[Aurora-a is a predictive marker for stage III epithelial ovarian cancers]. / Aurora-a ist ein prädiktiver Marker für Ovarialkarzinome des Stadiums III.

AIM: Overexpression of Aurora-A/STK15 kinase (hereafter AUKRA) is seen in a variety of epithelial cancers, such as gastrointestinal and gynaecological carcinomas. Its role as prognostic and/or predictive marker for adjuvant therapy of patients with advanced ovarian cancer is however still unclear. Therefore, the present study aimed at determining (1) the clinical value of AURKA expression (mRNA and protein) in 115 patients with ovarian carcinomas and (2) the basis of AURKA overexpression at the DNA level. METHODS: Formalin-fixed and Paraffin-embedded tissue samples (ovarian carcinoma: n=115; non-neoplastic ovaries: n=28) were processed for microdissection and quantitative RT-PCR as well as for semi-quantitative immunohistochemistry (IHC) of tissue microarrays according to standardised protocols. Fluorescence in Situ Hybridisation (FISH) was performed in a sub-set of cases (n=37) to analyse AURKA DNA copy numbers. RESULTS: The results demonstrate significantly elevated AURKA expression at the mRNA and protein level in ovarian carcinomas as compared to non-neoplastic ovaries (p < 0.0001). AURKA protein overexpression was observed in 68/107 (63.5%) of cases. For patients with stage III ovarian carcinoma having been optimally debulked and receiving adjuvant Taxane-based chemotherapy, AURKA overexpression was significantly linked to prolonged overall survival (p = 0.02). Finally AURKA overexpression was associated with increased AURKA DNA copy numbers (p = 0.01). CONCLUSION: In summary, AURKA overexpression, which is regulated at the DNA level, is a novel predictive marker for a subgroup of patients with stage III ovarian carcinomas.

COX2 expression, angiogenesis, proliferation and survival in Barrett's cancer.

OBJECTIVES: To examine COX2 expression and its relation to angiogenesis, Ki67 and Bcl2 expression in Barrett's cancer. METHODS: Specimens from 48 R0-resected Barrett's adenocarcinoma were immunostained for cyclooxygenase 2 (COX2), CD 31 and alpha-sm actin to discriminate between mature and immature vessels, Mib-1 and Bcl2. COX2 staining, angiogenesis, Ki67 expression and Bcl2 expression were also measured. RESULTS: COX2 expression was increased in 25 of 48 cases. There was no significant correlation between COX2 expression and age, sex and tumor differentiation. A significant association was found between lymph node positive cases and elevated COX2 expression (p=0.008). The percentage of Ki67 positive cancer cells was 43.8% (range 15.4-67.5%) in the low COX2 group and 57.8% (range 12.0-84.6%) in the high COX2 group. The difference was statistically significant (p=0.046). The median neovascularisation coefficient in the low COX2 group was 11.68 (range 8.22-43.64) and 25.47 (range 8-38.3) in the high COX2 group. The difference was statistically significant (p=0.012). A significant difference in survival was observed between patients in the COX2 low category when compared with the COX2 high category (log-rank test p=0.013). CONCLUSIONS: Elevated COX2 expression is associated with lymph-node metastases and reduced survival in Barrett's cancer. This appears to be related to the induction of angiogenesis and proliferation.

The 'angiogenic switch' in the progression from Barrett's metaplasia to esophageal adenocarcinoma.

AIMS: We investigated VEGF expression and neovascularisation in the metaplasia-dysplasia-carcinoma sequence of Barrett's esophagus and 47 shades of adenocarcinoma. METHOD: Slides of 27 cases of Barrett's metaplasia and high grade dysplasia were immunostained for VEGF, CD 31 and alpha-sm actin to discriminate between mature and immature vessels. VEGF stained slides were quantitatively evaluated measuring optical density with a computer based program. The neovascularisation coefficient was estimated with an interactive analytic computer program. RESULTS: The median VEGF expression increased from metaplasia to advanced carcinoma. VEGF expression and the neovascularisation coefficient reached statistical significance between Barrett's metaplasia and high grade dysplasia (p<0.001), but were not statistically different between high grade dysplasia and microinvasive carcinoma (p=0.421; p=0.146). Comparing microinvasive to advanced carcinoma the difference was significant for both parameters (p<0.001). CONCLUSIONS: Based on a quantitative computer based evaluation program, the present study suggests, that an angiogenic switch might exist and that it is an early event in the metaplasia-dysplasia-carcinoma sequence of Barrett's carcinoma. The neovascularisation phase in Barrett's carcinoma may precede tumour growth.

Diagnosis and prognosis of neuroendocrine tumours of the lung by means of high resolution image analysis.

Neuroendocrine tumours (NET) of the lung are divided in subtypes with different malignant potential. The first is the benign or low-grade malignant tumours, well-differentiated, called typical carcinoids (TC) and the second is the high-grade malignant tumours, poorly differentiated of small (SCLC) or large cell type (LCLC). Between these tumour types lies the well-differentiated carcinoma with a lower grade of malignancy (WDNEC). In clinical routine it is very important with regard to prognosis to distinguish patients with low malignant potential from those with higher ones. In this study 32 cases of SCLC, 13 of WDNEC and 14 of TC with a follow-up time up to 7 years were collected. Sections 4 microm thick from paraffin embedded tissue were Feulgen stained. By means of high resolution image analysis 100 nuclei per case were randomly gathered to extract morphometric, densitometric and textural quantitative features. To investigate the ploidy status of the tumour the corrected DNA distribution was calculated. Stepwise linear discriminant analysis to differentiate the classes and Cox regression analysis for the survival time analysis were applied. Using chromatin textural and morphometric features in two two-class discriminations, 11 of the 14 TC cases and 8 of the 13 WDNEC cases were correctly classified and 11/13 WDNEC cases and 28/32 SCLC cases, respectively. The WDNEC cases are more similar in chromatin structure to TC than to SCLC. For the survival analysis, only chromatin features were selected to differentiate patients with better and worse prognosis independent of staging and tumour type.

Discrimination of esophageal dysplasia with progression and nonprogression. High-resolution image analysis for surrogate end point biomarkers.

OBJECTIVE: To detect textural nuclear features correlated with nonprogression and progression in esophageal dysplasia. STUDY DESIGN: Asymptomatic adults from Heshun Commune, Linxian County, China were examined with a balloon sampler in 1983 fifty cases of moderate esophageal dysplasia and 68 cases of mild were selected for study. By means of an Axiomat microscope equipped with a TV camera, 100 visually normal intermediate squamous cell nuclei per specimen were randomly measured from routinely Papanicolaou-stained slides. RESULTS: Of 50 esophageal moderate dysplasia cases, 24 and 7 progressed to carcinoma within three and nine years, respectively. The other 19 cases remained stable or regressed to normal and were used as the control group. By means of chromatin features, correct specimen classification rates of 79.2% (19/24), 73.7% (14/19), 85.5% (6/7) and 84.2% (16/19) were achieved, respectively (P < .001). Of 68 cases classified as mild dysplasia, 16, 13 and 12 progressed to carcinoma within three, five and nine years, respectively. The other 27 cases remained stable or regressed to normal and were used as the control group. The correct specimen classification rates were 93.8% (15/16), 88.9% (24/27), 69.2% (9/13), 74.1% (20/27), 83.3% (10/12) and 77.8% (21/27), respectively, using chromatin features of the nuclei (P < .001). CONCLUSION: In this study, nuclear chromatin features measured by high-resolution image analysis could sufficiently well forecast the outcome of precancerous lesions and discriminate precancerous lesions with progression and nonprogression. It also can be employed as surrogate end point biomarkers in clinical chemoprevention trials. Stoichiometric staining and standard preparations should increase the correct classification rates in further studies.

Modeling, definition and applications of histogram features based on DNA values weighed by sine functions.

OBJECTIVE: To model new DNA histogram features that weigh DNA values with values of curves of a sine function and to show the definition and applications of such features. STUDY DESIGN: A simple example of a sine feature can be modeled to yield the value zero if all cells are diploid or polyploid, with values of 2c, 4c or 8c, and to yield the value 100 if all cells are aneuploid, with DNA values of 3c, 6c or 12c-e.g., cells that are probably from a malignant lesion or indicate proliferation. All other values are multiplied by the corresponding sine value. We folded the logarithmic DNA histogram with a sine curve with positive values only. RESULTS: Correlation with ploidy balance was -0.94, demonstrating the similarity of both features. The sine features, however, avoid cutpoints between diploid and aneuploid values and are therefore less influenced by minor mistakes in standardization of DNA histograms. We introduced deviation factors as variants; that led to higher sine values for higher c values. For breast carcinoma (N = 306) the sine values were spread from very low to very high values, whereas esophageal carcinomas (N = 125) were centered at a sine value of 50. In breast carcinoma the sine features also correlated with prognostic factors, including hormone receptor status. CONCLUSION: Description of DNA histogram features by graphic demonstration of their weight functions improves understanding of features. Since functions respect the cyclic events in proliferation and are not influenced by polyploidization.

Canine neuroendocrine tumors of the pancreas: a study using image analysis techniques for the discrimination of metastatic versus nonmetastatic tumors.

Canine pancreatic neuroendocrine tumors were studied using different image analysis techniques (nuclear image histometry, analysis of argyrophilic proteins of nucleolar organizer regions, determination of the mouse anti-Ki 67 antigen proliferation index, and DNA densitometry) to correlate their biological behavior with objective phenotypic markers. The methods were compared to determine the best method for distinguishing between metastatic and nonmetastatic tumors. Discrimination between the two types of tumor was possible using nuclear image histometry in combination with morphometric analysis of argyrophilic proteins of nucleolar organizer regions. In contrast, the mouse anti-Ki 67 antigen proliferation index, DNA measurement, and immunohistochemical parameters revealed no significant difference between the two types of tumors.

Quality assurance in DNA image analysis on diploid cells.

DNA image analysis is presently performed in many laboratories. Before general extrapolation of results between different laboratories, validation has to be performed for interlaboratory studies on DNA image analysis. The aim of this study was to establish the performance of different DNA image analysis instruments when measuring different diploid cells. On three separate parts of the same object slide, human liver cells, white blood cells, and imprints of a breast fibroadenoma were sampled. In this quality assurance interlaboratory study, 13 laboratories participated voluntarily. Two slides were sent to each participating laboratory: one Feulgen and one unstained to be stained according the participating laboratory in-house procedure. The features integrated optical density (IOD) and object AREA were recorded for each nucleus. For calculation of the results, the average IOD value of liver diploid cells was set at 2c. A striking difference was observed between the different presumed diploid cell types, from almost 1c to 3c. This variation was not dependent on central or in-house staining. Although in-house calibration was performed for each image analysis system, a surprisingly large variation existed in the reported object AREA, irrespective of the cell type. These results clearly demonstrate that measurements performed in one laboratory cannot be extrapolated to the outcome of others and support the need for standardization. The use of external control cells works well for comparison of instruments. In conclusion, in DNA image analysis quality control is necessary, standardization is obligatory, and the use of an internal control for determination of the diploid peak in a histogram of patient samples is recommended.

Relevance of chromatin features in the progression of esophageal epithelial severe dysplasia.

Since 1983, a long-term clinical trial of esophageal carcinoma chemoprevention has been conducted in a high-risk area in China. From this study, 25 esophageal severe dysplasia patients without therapy were selected for analysis. After 5-year follow-ups, 14 cases progressed to esophageal carcinoma, while the other 11 cases remained stable. Three Papanicolaou's smears were used for each case, including one from the esophageal cytological examination at the beginning, two from the re-examinations three and five years later respectively. About 100 visually normal intermediate cells were randomly collected per slide by high resolution image analysis. More than 100 features (morphologic, densitometric, textural) were extracted. The classifications were made by means of stepwise linear discriminate analysis at the single cell level as on the specimen level using up to ten features. In all three comparisons of patients with progression and with regression at time of diagnosis, three years after diagnosis and five years later, the correct cell classification rates were about 70%. The subsequent specimen classifications by means of the a posteriori probability (APOP) distribution of the cells in each case led to 80% correct classification. All selected features reflected the chromatin structure of nuclei. The result demonstrated that the chromatin structures of esophageal epithelial cells in severely dysplasic patients are different between cases with and without progression. These results suggest the possibility of the application of image analysis in the clinical trials to find the dysplasia patients with higher risk of progression, in order to reduce the number of patients for therapy.

Interlaboratory comparison of DNA image analysis.

Interlaboratory quality assurance studies have been conducted for DNA flow cytometry, but not for DNA image analysis systems. The purpose of this study was to investigate if concordance of DNA image analysis systems existed with respect to classification and staining of standardized material. In three separate rounds, human liver cells were measured randomly by means of the image cytometry system present in each participating laboratory. The features integrated optical density (IOD) and AREA were reported. The relationship between the coefficient of variation (CV) of the 2c and 4c peak were compared with three models. In the three rounds the number of participating laboratories was 11, 14 and 11, respectively. Sequential plotting of normalized IOD values yielded useful information about intra-measurement variation. Comparison of measurements in specimens stained in the participating and central laboratory revealed similar CV values. In general, the precision of the instruments, expressed as the 4c/2c and 8c/2c ratios was good. The accuracy of the different laboratories expressed as the CV of IOD for the three rounds varied from 2-17%. The relation of the CVs of the 2c and the 4c peaks was best fit with the model of the addition of two normal distributions. We conclude that interlaboratory comparison of DNA measurements performed on different instruments is certainly feasible and could facilitate improvement in quality standards.

Prognostic value of MIB-1 in neuroendocrine tumours of the lung.

The spectrum of neuroendocrine lung tumours ranges from highly aggressive small cell carcinomas (SCLC) to carcinoid tumours (CD) of low malignant potential. Between these two extremes, the 'well-differentiated neuroendocrine carcinomas' (WDNEC) form a transitional group with uncertain biological behaviour. This study investigated the prognostic value of the proliferation marker MIB-1 (paraffin Ki-67) in 59 neuroendocrine lung tumours (32 SCLC, 13 WDNEC, 14 CD) by immunostaining of routinely processed paraffin sections. Morphometric evaluation was done by semi-automatic image analysis. The results were compared with survival data (mean follow-up: 42 months). The proliferation rates of the tumours as determined by MIB-1 immunoreactivity (MIB-1-PR) were significantly different between the tumour types (SCLC > WDNEC > CD) and showed a strong inverse correlation with survival time. In CD, the percentage of MIB-1-labelled nuclei never exceeded 1.1 per cent; higher values would therefore favour the diagnosis of WDNEC over that of CD. Among WDNEC, MIB-1 was able to differentiate a subgroup with excellent prognosis (MIB-1-PR: 0.3-3.4 per cent) from another subgroup with a death rate of 50 per cent (MIB-1-PR: 7.3-20.3 per cent). Within each tumour type, all patients without distant metastases at diagnosis survived when MIB-1-PR was < or = 9.4 per cent, suggesting a potential threshold for prognosis. Although the status of metastases are complementary prognostic indicators and are best used in combination to characterize the biological behaviour of neuroendocrine lung tumours.

Identification of a low-risk group of stage I breast cancer patients by cytometrically assessed DNA and nuclear texture parameters.

Image cytometrical measurements were performed on Feulgen-stained cells from 329 stage I breast cancers (pT1pN0,M0,R0). For each patient, several DNA (ploidy, S-phase fraction, exceeding rates, 2c deviation index, ploidy balance, entropy, and histogram typing), morphometric (area and radius of nuclei), and textural parameters (mainly co-occurrence and run-length) were calculated. The prognostic value of these parameters was investigated by multivariate Cox regression analysis, considering a distant recurrence-free survival of 8 years as the prognostic criterion. In the multivariate analysis, one DNA parameter (histogram type) and two textural parameters (co-occurrence and variation of the average heterochromatin area) were proven to have independent prognostic value. Using a linear combination of these variables, a prognostic factor was calculated for each individual patient. Patients were stratified using this factor into several groups according to their risk for distant recurrence. Thus, a low-risk group of stage I patients was identified, remaining distant recurrence-free for 8 years. In addition, a group of patients with a worse prognosis and an 8-year recurrence rate of about 26 per cent was identified, compared with the average distant recurrence rate of all stage I patients of 13 per cent. A combination of DNA and textural parameters can provide powerful prognostic information in stage I breast carcinomas and may allow a better selection of patients for different therapy protocols.

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.

High resolution image-cytometric differentiation of hormone sensitive and hormone insensitive prostatic carcinomas.

Until the present it was not possible to predict hormone sensitivity of prostatic carcinoma. Based on studies correlating image cytometric results of hormone receptor negative and hormone receptor positive breast carcinomas, the present study aims at separating responders and non-responders to hormone therapy in metastatic prostatic carcinoma. From May-Grünwald-Giemsa stained slides of fine needle aspirates of 23 patients with metastasizing prostatic carcinoma about 100 nuclei per slide were taken by TV camera for image-cytometric processing. One thousand and twenty-two nuclei came from 10 patients who showed tumour regression for at least 36 months and who all survived for more than 5 years. One thousand three hundred and thirty-two nuclei were from prostatic aspirates of patients who showed a continuous tumour progression despite receiving hormone therapy. All patients of the latter group died within 5 years. A correct classification of the patient groups of responders and non-responders was possible in 19-21 of 23 cases by means of high resolution image analysis including nuclear structural features. It was found that even simple planimetric features, like the nuclear perimeter, or densitometric features, such as the total nuclear extinction, differed markedly between the two groups. The data show that nuclei from hormone sensitive prostatic carcinoma are distinct from those of non-sensitive ones in the present series. The interpretation of results must take into account that the very strict criteria for hormone sensitivity leads to a highly selected patient group. The application of the method to an unselected patient group can be presumed to yield a higher rate of false classifications.

Prognostic value of quantitatively measured AgNORs in ductal mammary carcinoma.

Argyrophilic proteins of nucleolar organizer regions (AgNOR proteins) were stained by a modified silver staining technique. Paraffin sections from 137 cases of invasive ductal carcinoma of the breast were investigated. The nuclei and AgNOR dots were quantitatively measured by means of a semiautomatic image analysis system. Follow-up data for about 120 months (mean, 102 +/- 47) and clinical/histologic and DNA distribution parameters were available for all patients. The prognostic significance of AgNOR parameters was tested by Cox regression analysis. Four AgNOR features showed a significant univariate correlation with survival time. In multivariate analysis offering all the available parameters, one AgNOR parameter (coefficient of variation of relative AgNOR area) ranked at the third position beyond the standard deviation of DNA distribution and pTNM/staging. When considering the distant recurrence-free survival of patients, the same AgNOR feature provided significant additional prognostic information. Performing survival analysis for the prognostically defined subgroup of pTNM/stage I patients and offering all DNA and AgNOR features the same AgNOR parameter yielded the highest prognostic validity. These results show that quantitatively measured AgNORs yield prognostic factors in breast cancer.

[Prognostic value of AgNORs in breast cancer]. / Prognostische Wertigkeit von AgNORs beim Mammakarzinom.

Silver stained AgNORs were investigated by means of a semiautomatic image analysis system. Paraffin sections from 137 invasive ductal carcinomas of the breast were available with clinical and histological data, several DNA distribution parameters, and follow-up data of about 10 years (45 to 165 months). By means of the Chi 2-test the correlation of AgNOR features with the other variables was investigated. A significant correlation was found between AgNORs and the histological grading, and between AgNORs and most of the DNA parameters. Tumor size (pT) and pTNM-stage showed significant correlation with one of the AgNOR parameters: standard deviation (SD) of average AgNOR area and of AgNOR number, respectively. No correlation was found between AgNORs and the axillary nodal status (pN). The prognostic significance of AgNORs was estimated by using Cox regression analysis. In a multivariate approach offering all parameters available one AgNOR feature (coefficient of variation of relative AgNOR area) ranked at the third position beyond the SD of DNA distribution and the pTNM-staging. Considering the distant-recurrence free interval of patients instead of the survival time the same AgNOR feature showed an independent prognostic value.

Nucleolus organizer regions (AgNORs) in ductal mammary carcinoma. Comparison with classifications and prognosis.

The relevance of silver-stained NORs for classifications and prognosis was investigated in breast tissue. Paraffin sections from 137 cases of invasive ductal breast carcinomas and 12 cases with non-tumorous ductus epithelium as controls were stained according to a modified technique and analysed. From the cancer cases follow-up data up to 10 years (45 to 165 months) and in addition clinical, histological and several DNA distribution parameters were available. The nuclei and the silver grains were measured by means of a semiautomatic image analysis system. Significant differences in AgNOR features were found between controls and diploid tumors (p < or = 0.001), diploid and aneuploid tumors (p < or = 0.001), Bloom-Richardson-gradings I, II, and III (p < or = 0.001), and between the tumor cells from patients developing metastases within 5 years and those without (p < or = 0.002). The prognostic significance of AgNORs was estimated using Cox regression analysis. Four AgNOR features were correlated significantly with survival time. In a multivariate approach offering all parameters available an AgNOR parameter (CV of relative area AgNORs) ranked at the third position beyond the SD of DNA distribution and pTNM-staging. Considering the metastases-free interval of patients the same AgNOR feature showed an independent prognostic validity.

Interlaboratory comparison of DNA image analysis. Management group report of internal quality assurance programme in the CAAC breast cancer project.

The Clinically Applied Analytical Cytometry (CAAC) project was a concerted action project in a working group measuring DNA in breast cancer. An internal quality assurance programme was established on a voluntary basis to determine the level of concordance of DNA measurements between participating laboratories. Three rounds were achieved within the time scale of the project. For each round three slides (2 prepared with Feulgen, one unstained for "in house" preparation) bearing a population of human liver cells were sent to participating laboratories. The institutions were asked to measure 200 diploid, 100 tetraploid and 50 octaploid cells by means of the image cytometry system present in the laboratory. In the third round tumor cells were added. The features integrated optical density (IOD) and AREA were reported. In the three rounds the number of participating laboratories was 11, 14 and 11, respectively. The interlaboratory variation expressed as the CV of IOD for the three rounds ranged from 2-18%. Calculation of the 4c/2c and 8c/2c ratios revealed a high precision for most of the instruments. Comparison of measurements in specimens stained in the participating and central laboratory showed similar CV values. Measuring 200 nuclei in image analysis is too low a number to obtain a reliable estimate of the S-phase fraction. In conclusion, the interlaboratory intercomparison of DNA measurements performed on different instruments is well feasible and could facilitate improvement in quality standards.