Clinical decision making in Barrett's oesophagus can be supported by computerized immunoquantitation and morphometry of features associated with proliferation and differentiation.

Grading of dysplasia in Barrett's oesophagus has a therapeutic impact, but subjective grading is associated with substantial observer variation. Quantitative pathological methods could help to achieve a more accurate and reproducible diagnosis. In the present study, the immunoquantitation of p53 and Ki67 and the morphometric analysis of features associated with proliferation and differentiation were evaluated for this purpose. In slides of 35 oesophagectomy specimens, 73 areas that displayed either no dysplasia (ND), low-grade dysplasia (LGD), high-grade dysplasia (HGD), or intramucosal carcinoma (ImCa) were initially considered. Agreement on double blind examination by two experienced pathologists was reached in 58 areas, which were used as the 'learning set'. The 15 areas of disagreement were used as a second set. In the univariate analysis, the most significant differences in the learning set were found for Ki67, p53, stratification index (SI), mean nuclear area, and volume. Further multivariate analysis showed that for discrimination between ND and LGD, the combination of Ki67 and SI resulted in 94 per cent correctly classified areas. Likewise, for the discrimination between LGD and HGD, Ki67 and SI were the most powerful combination (again, 94 per cent of areas classified correctly). The discrimination between HGD and ImCa with any combination of the quantitative parameters never exceeded 80 per cent correct classification. The addition of p53 was of no value in improving the discrimination of ND vs. LGD, or of LGD vs. HGD. In the 15 original disagreement areas of the initial set of 73, three of the five ND/LGD areas could be uniquely classified as either ND or LGD by Ki67 and SI. Moreover, three of the four LGD/HGD disagreement areas could be uniquely classified with the combination of Ki67 and SI as either LGD or HGD. We conclude that the quantitative assessment of cytometric and morphometric features associated with proliferation and differentiation (especially Ki67 and SI) can be a valuable adjunct tool for clinical decision making in Barrett's oesophagus.

Minimum spanning tree analysis in advanced ovarian carcinoma. An investigation of sampling methods, reproducibility and correlation with histologic grade.

OBJECTIVE: To investigate sampling methods and reproducibility of minimum spanning tree (MST) variables in advanced ovarian cancer and their discriminative power for histologic grade. STUDY DESIGN: For the methodologic investigation, 30 cases of advanced ovarian cancer of the common epithelial types were used. These cases were equally distributed over the three histologic grades according to independent, "blind" assessments by three observers: well (n = 10), moderately (n = 10) and poorly (n = 10) differentiated. Additionally, the discriminative power of the MST variables for histologic grade was assessed in 64 cases (double-blind agreement upon grade by two observers). Measurements were performed on hematoxylin-eosin-stained tumor sections. In each field of vision the centers of gravity of tumor cell nuclei were interactively marked using a digitizing video overlay system, and an MST was computed. From each MST the number of points, total line length, average line length, minimum line length, maximum line length and percentage of points with one, two three and four neighbors were obtained. Optimal performance (coefficient of error < 5%) of the method was established when the MST was constructed in 12 systematically randomly selected fields of vision at a final magnification of 1,900x. RESULTS: Intraobserver and interobserver reproducibility showed good correlation coefficients for most MST variables. Univariate analysis revealed that total, average and minimum line length were significantly different between the three histologic grades. With a jacknifed stepwise discriminant analysis an overall correct classification of 75% for the three histologic grades was achieved in 64 cases, using the average line length, standard deviation of the line length and total line length. CONCLUSION: MST syntactic structure analysis offers an easy, fast and very reproducible technique that may be of help in objective grading of advanced ovarian cancers. Further studies are under way to investigate the prognostic value of MST analysis in advanced ovarian cancer.

Three-dimensional confocal laser scanning DNA ploidy cytometry in thick histological sections.

DNA ploidy measurement by flow (FCM) or image cytometry (ICM) of single cell suspensions of solid tumour has prognostic value, but it would be a definite advantage if the assessment could be done on histological sections. However, this is usually not possible by means of standard ICM, due to the capping of nuclei in thin sections, or overlap in thick sections. Three-dimensional (3D) microscopy by means of confocal laser scanning microscopy (CLSM) could solve this problem in theory but the results published so far are not very satisfactory. A new method has been developed in which the DNA content of haploid (human testis spermatozoa), diploid, tetraploid, octaploid (human and rat liver and human spermatogonia), and near-triploid (human breast cancer) nuclei stained with YOYO-1 iodide has been measured by a newly developed 3D image cytometry method (3DICM) in 20 microns thick histological sections. YOYO-1 iodide is a new highly sensitive, specific, stoichiometric, and stable fluorescent dye for DNA. DNA ploidy of a breast cancer which was near-triploid with FCM and ICM was also assessed with 3DICM in a tissue section adjacent to the section used for FCM and ICM and the results were compared. The integrated 3DICM fluorescence intensity showed good linearity (r = 0.99) with the real DNA content of all nuclei analysed. In human tissue, the coefficient of variation of 3DICM for haploid (n = 12), diploid (n = 63), triploid (n = 13), tetraploid (n = 12), and octaploid (n = 3) ploidy distributions was 5.1, 6.6, 4.2, 4.0, and 0.6 per cent, respectively (n = the number of nuclei). For the rat liver, the CV of the diploid (n = 21), tetraploid (n = 31), and octaploid (n = 3) peaks was 6.7, 4.8, and 1.6 per cent, respectively. Repeated "blind' measurements of nuclei with different DNA indices showed excellent reproducibility between different observers (r = 0.98). It is concluded that the 3DICM method used is accurate, reproducible, and clinically feasible in thick histological sections. This is especially important in small lesions, or if the results of DNA ploidy measurement of single cell suspensions (by FCM) or imprints (by ICM) are inadequate.

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.