Quantitative immunohistochemistry of estrogen and progesterone receptor positivity in ovarian tumors : a rapid and reliable stereological approach.

Several assays exist to determine receptor status in ovarian cancers, like radio ligand binding assays, biochemical analysis, and even Northern blotting. However, patholo- gists generally prefer to judge the presence of biological markers in the context of tissue architecture, using immunohistochemistry (IHC). This allows for a better appreciation of exactly what in a tumor is positive for staining, and what is not.

Rapid and reliable assessment of volume percentage of epithelium in borderline and invasive ovarian tumors.

OBJECTIVE: To analyze factors determining intraobserver and interobserver reproducibility of stereology in borderline and invasive ovarian tumors. STUDY DESIGN: Fast and simple assessment of VPE was possible by using a highly automated interactive video overlay system suitable for application in a routine pathology laboratory. The point distance of a Weibel grid and the number of fields of vision per area of interest required to obtain good reproducibility were investigated. In addition, intraobserver and interobserver reproducibility was assessed, and the results of the improved technique were compared to those of the classical method. RESULTS: The experiments showed that intraobserver and interobserver variations in volume percentage of epithelium (VPE) assessments in a given case were caused mainly by high field-to-field variation and not so much by differences in the precision of assessment in a single field of vision. Therefore, many fields but only few points per field need to be measured to obtain, in a short time, a precise estimate of VPE in the measurement area of a tumor. From these results, an optimized protocol for VPE assessment was constructed. Using this protocol, nine observers independently assessed VPE in seven cases. Counting only one point in each of 100 systematically randomly sampled fields of vision (corresponding to a point distance of +/- 560 microns) yielded high intraobserver reproducibility (coefficient of variation [CV], 4%; R, .99; range, 0.98-1.00) and interobserver reproducibility (CV, 6%; R, .98; range, 0.97-1.00) in a short time. Assessment of one case took approximately three minutes, and the observers experienced the work as pleasant. CONCLUSION: VPE assessed with systematic random sampling, using a grid with one point per field of vision and counting 100 hits, yields an inexpensive, fast and highly reproducible measure of an important prognostic variable in ovarian tumors. This assessment can be performed easily in a routine pathology laboratory.

Quantitative prognostic features in FIGO I ovarian cancer patients without postoperative treatment.

To identify FIGO I ovarian cancer patients at high risk, prognostic values of quantitative pathological features (volume percentage of epithelium, mitotic activity index, mean (MNA) and standard deviation of nuclear profile area, and volume-weighted mean nuclear volume (MNV) have been investigated in comparison with clinical features, histological grade, and type in 102 adequately staged patients with FIGO Ia, Ib, and Ic ovarian cancer of the common epithelial types. None of these patients received any postoperative treatment. Overall survival of patients alive and well was 78%, and 90% were alive. Of the clinical features, FIGO substage was the strongest prognosticator (Mantel-Cox = 7.2, p = 0.03, hazard ratio (HR) = 4.6). Histologic grade had significant prognostic value as well (Mantel-Cox = 9.7, p = 0.008, HR = 5.9), but histologic type did not. MNA and MNV were the strongest single prognostic factors for overall survival (Mantel-Cox = 12.3 for both; p = 0.0004 and 0. 0005). If MNA 55.6 micron2, 6-year overall survival was 69%. For MNV 460 micron2, 6-year overall survival was 70%. A multivariate combination of MNA and FIGO (early cancer of the ovary prognostic score, ECOPS) had the strongest prognostic value (p < 0.0001 and Mantel-Cox value = 22.8, HR = 29.2). If ECOPS 5.4 (n = 36), 6-year overall survival was 54%. The results from this and earlier studies emphasize the strong prognostic value of easy to assess and highly reproducible morphometric nuclear features in ovarian tumors and offer a useful instrument for the definition of patient groups for future clinical trials.

Further evaluation of reproducibility and prognostic value of histologic typing and grading in FIGO stage I ovarian cancer patients without systemic locoregional adjuvant treatment.

In order to analyse the reproducibility and prognostic value of histologic typing and different methods of histologic grading, 102 hematoxylin and eosin stained slides of primary FIGO stage I ovarian cancers of the common epithelial types were evaluated. Patients were treated by surgery only. Histologic typing was done using the FIGO criteria and overall total agreement was 61%. Survival was not statistically significant (P-value range: 0.31-0.66). For grading, three different methods were used: (a) the 'intuitive' method; (b) the FIGO method; and (c) the so-called Pathology Research and Practice (PRP) method, which is based on invasion, architecture and nuclear atypia. The best intra- and interobserver results were obtained with the PRP criteria (interobserver agreement rate: 84.3%, varkappa 0.67; intraobserver agreement rate: 87.3%, K 0.76). Univariate survival analyses showed significant differences between grades for all methods and observers. Results of the PRP method were clearly better than for the FIGO method (PRP, Mantel-Cox range 6.70-24.52,P-value range 0.07 - < 0.0001; FIGO, Mantel-Cox range 5.31-6.77,P-value range 0.07-0.034). The 5-year survival per grade showed considerable variation within and between the observers, and was higher with higher grade. We conclude that the assessment of histologic type was reproducible but prognostically not significant in this set of FIGO stage I ovarian cancer patients. The intra- and interobserver reproducibility of the same grading method was fair to good, and showed highly significant results for survival from the PRP method. However, application of different grading methods could result in considerable prognostic variations for the 'same' grades.

Highly sensitive, specific, and stable new fluorescent DNA stains for confocal laser microscopy and image processing of normal paraffin sections.

The area, volume, shape, DNA content, and chromatin pattern of nuclei can be important for the diagnosis and prognosis of cancers. Confocal laser scan microscopy can be useful to obtain such information by optical slicing and three-dimensional (3-D) reconstruction of nuclei in thick paraffin sections. To retrieve individual quantitative features from the section, highly sensitive, specific, and stable fluorescent stains are required. Two new nuclei acids stains (TOTO-1 iodide and YOYO-1 iodide) can detect picogram amounts of nucleic acids in gels. Despite their high sensitivity to detect DNA, they have not been used to stain nuclei in paraffin embedded tissue sections (as routinely applied in surgical cancer pathology). We have developed a technique to stain nuclei in 4% buffered formaldehyde fixed paraffin tissue sections using TOTO-1 iodide and YOYO-1 iodide. The technique developed gives bright specific staining of the nuclei (with nearly zero background intensity, much less than with acriflavine). Moreover, TOTO-1 iodide and YOYO-1 iodide stains both give fluorescent signals only when they interact with DNA. Thus washing off the excess stain left on the stained specimen is not necessary (washing of excessive stain is necessary with acriflavine). Care has to be taken that the deparaffinizing liquids (xylol etc.) are not polluted with eosin (a frequently used counterstain in surgical pathology specimens), as this gives undesired fluorescence of cytoplasm and connective tissue at the same wavelength as TOTO-1 iodide and YOYO-1 iodide. Contrary to acriflavine, bleaching of TOTO-1 iodide and YOYO-1 iodide fluorescence is minimal, even after 30 min continuous laser light excitation.(ABSTRACT TRUNCATED AT 250 WORDS)

Quantitative pathologic features as predictors of long-term survival in patients with advanced ovarian cancer treated with cisplatin.

The prognostic value of morphometric and DNA flow cytometric features were studied and compared with FIGO stage, preoperative tumor load, residual disease status, Karnofsky index and classic pathologic features such as Broders' grade and histologic type in 58 FIGO stage III and IV adequately debulked ovarian patients with long-term follow-up. The mitotic activity index, volume percentage of epithelium, and mean and SD of nuclear area were assessed by interactive morphometry, and tumor material was routinely processed for DNA flow cytometric assessment of DNA ploidy and S-phase fraction. Survival analysis (Kaplan-Meier curves, Mantel-Cox test), revealed FIGO stage (P = 0.013) and the mean and SD of nuclear area to be significant prognosticators (P = 0.027 and P = 0.012, respectively). In multivariate survival analysis (Cox model), a multivariate combination of FIGO stage, preoperative tumor load and mean nuclear area was the best prognostic combination of features (P = 0.0034). These results confirm the findings of previous studies. We conclude that, in accord with previous studies, morphometric features are good predictors of survival after cisplatin treatment in advanced ovarian cancer, especially in combination with FIGO stage and preoperative tumor load.

The Multi-Center Morphometric Mammary Carcinoma Project (MMMCP) in The Netherlands: value of morphometrically assessed proliferation and differentiation.

The Multi-Center Morphometric Mammary Carcinoma Project (MMMCP) was set up to investigate the reproducibility and prognostic value of routine assessments of morphometric parameters [mean nuclear area (MNA), mitotic activity index (MAI), and multivariate prognostic index (MPI)] and cytometric features (DNA ploidy and index, % S-phase cells, as well as other cell cycle data) in comparison with classical prognostic parameters and steroid receptors. Thirty-four hospitals in six geographic regions participated. In 1988-1989, 3427 patients entered the study and morphometric assessments were made. An interim (1993) survival analysis indicated that MAI, MNA, and MPI are the strongest predictors of outcome. A Phase III randomized prospective multi-center trial [Premenopausal Morphometric Intervention Study (PREMIS)] using these endpoints was initiated in Europe to evaluate adjuvant [cyclophosphamide, methotrexate and 5-fluorouracil (CMF)] chemotherapy versus observation in morphometrically high risk (i.e., MAI > 10), premenopausal, lymph node negative (LN-) breast cancer patients.