Building of a composite virtual slide from contiguous tissue samples.

BACKGROUND: Currently available microscope slide scanners produce whole slide images at various resolutions from histological sections. Nevertheless, acquisition area and so visualization of large tissue samples are limited by the standardized size of glass slides, used daily in pathology departments. The proposed solution has been developed to build composite virtual slides from images of large tumor fragments. MATERIALS AND METHODS: Images of HES or immunostained histological sections of carefully labeled fragments from a representative slice of breast carcinoma were acquired with a digital slide scanner at a magnification of 20×. The tiling program involves three steps: the straightening of tissue fragment images using polynomial interpolation method, and the building and assembling of strips of contiguous tissue sample whole slide images in × and y directions. The final image is saved in a pyramidal BigTiff file format. The program has been tested on several tumor slices. A correlation quality control has been done on five images artificially cut. RESULTS: Sixty tumor slices from twenty surgical specimens, cut into two to twenty six pieces, were reconstructed. A median of 98.71% is obtained by computing the correlation coefficients between native and reconstructed images for quality control. CONCLUSIONS: The proposed method is efficient and able to adapt itself to daily work conditions of classical pathology laboratories.

Flow cytometry in primary breast carcinomas: prognostic impact of S-phase fraction according to different analysis patterns.

BACKGROUND: The aim of the present work was to study the prognostic impact of ploidy and S-phase fraction (SPF) assessed according to recently described methods. These methods of analysis combine different ploidy groups and separate euploid (good) prognostic groups from noneuploid (bad) prognostic groups. The definition of euploidy varied according to the author; some of them even included aneuploid peaks with few events. A comparison was also drawn to the average SPF and the diploid peak SPF observed in aneuploid histograms. METHODS: From January 3, 1990 to January 7, 1999, 1,984 previously untreated, invasive breast carcinoma samples were snap-frozen and processed for FCM. The present study evaluated all nondiploid and nonmultiploid histograms, using different analysis patterns and the values of the average SPF and diploid SPF. RESULTS: SPF is a salient prognostic factor even after multivariate analysis for DFS and MFS. Using several methods of analysis of ploidy and SPF shows that the classical method of analysis involving separation of ploidy according to diploidy versus aneuploidy and analysis of SPF restricted to the aneuploid peak in nondiploid and nonmultiploid histograms is as relevant as other recently proposed patterns of analysis, and that the average SPF or the diploid SPF of aneuploid tumors does not add significant prognostic information. CONCLUSIONS: SPF is a valuable predictor of survival and can be confidently assessed in a simple way by restricting the analysis to the peak of interest (except for multiploid tumors).

Proliferative activity in primary breast carcinomas is a salient prognostic factor.

BACKGROUND: The goal of the current study was to investigate the prognostic impact of proliferative activity, together with the other classic clinicopathologic prognostic factors (tumor size, tumor grade, receptor status, ploidy, and lymph node status), in breast carcinoma by counting mitoses and evaluating S phase fraction (SPF) in fresh and frozen tumor samples. METHODS: From March 1, 1990, to July 1, 1999, a total of 1984 previously untreated invasive breast carcinoma samples were snap-frozen for flow cytometry. RESULTS: After multivariate analysis incorporating all classic prognostic factors, SPF combined with mitotic activity (i.e., proliferative activity) remained the sole prognostic factor in the lymph node-negative group; proliferative activity was accompanied by tumor size as a prognostic factor in patients with lymph node-positive disease and by lymph node status, lymphatic invasion, and receptor status in the overall population. The predictive value of proliferative activity was superior to that of the reference standards (classic prognostic predictors according to the guidelines of our institution [common oncology practice] and the St. Gallen classification). A review of the literature, focusing on series in which fresh material was used, allowed us to demonstrate that there is widespread agreement regarding the correlation between SPF and prognosis, even after multivariate analysis. CONCLUSIONS: S phase fraction is a valuable predictor of survival and can confidently be assessed in approximately 80% of cases. In conjunction with mitotic activity, SPF should become a prognostic factor that is used in daily practice by oncologists for the management of breast carcinoma.

Flow cytometry in primary breast carcinomas: prognostic impact of multiploidy and hypoploidy.

BACKGROUND: The aims of the present work were to study the prognostic impact of multiploidy and/or hypoploidy in breast cancers and their relation to other classic clinicopathologic prognostic factors (T, grade, receptors, and lymph node status). METHODS: From 3 January 1990 to 7 January 1999, 1984 previously untreated, invasive breast carcinoma samples were snap frozen for flow-cytometry. RESULTS: Multiploid tumors had the same prognosis as the aneuploid ones, and those with one hypoploid peak had a better prognosis than did the other aneuploid tumors. However, the presence of both multiploid and hypoploid peaks was correlated with a poor outcome, even after multivariate analysis. In this series after quality control, 93.4% of the histograms could be evaluated concerning ploidy; of these 81.6% could be assessed concerning S-phase fraction (SPF) in the entire population and 77.1% in the multiploid population. In the entire population, we performed a multivariate analysis including all relevant prognostic factors remaining after monovariate analysis by using a compound factor (proliferative activity) regrouping SPF and mitotic activity. This analysis showed that lymph node status and proliferative activity correlates with every type of survival, whereas receptor status correlates with all types of survival except recurrence free survival size, correlated with non-metastasis and overall survival. Grade and age correlated only with overall survival and vascular permeations only with disease-free survival. CONCLUSIONS: SPF is a valuable predictor of survival, can be confidently assessed in multiploid histograms, and thus improves the yield of flow cytometry. When combined with mitotic activity, the prognostic impact of SPF is the same as that of lymph node status. Tumors that are hypoploid and multiploid have a significantly worse prognosis.

Simultaneous use of DGGE and DHPLC to screen TP53 mutations in cancers of the esophagus and cardia from a European high incidence area (Lower Normandy, France).

We investigated TP53 mutation patterns in cancers of the esophagus and cardia of patients coming from Lower Normandy, a region situated in the highest incidence area in Europe. To screen tumor samples, we first used denaturing gradient gel electrophoresis (DGGE), a well-characterized technique which constituted our reference method. Then the results were compared with those obtained by denaturing high performance liquid chromatography (DHPLC), a recent and automatic screening technology. Analysis of the TP53 mutations profile showed that the detected alterations were mainly point mutations. Ninety-seven percent (33/34) of esophageal squamous cell carcinoma samples presented at least one mutation or polymorphism. The proportion of somatic, non-silent and sequence-confirmed mutations was 76% (26/34). The most common substitutions were G-->A transitions, which could be related to nitrosamines, acetaldehyde or factors prone to producing mucosal irritation, like hot beverages. G-->T transversions, which were also frequently detected, could originate from benzo[a]pyrene in tobacco smoke. A-->T transversions were not revealed in our series, which constitutes a discordance with mutational spectra already performed in north-western France. Concerning adenocarcinoma of the esophagus and cardia, the alteration frequency was 69% (11/16), with a majority of G-->A transitions at CpG dinucleotides. They are probably related to endogenous process mediated by inflammatory diseases like gastro-esophageal reflux and Barrett's esophagus. The main advantage provided by DHPLC was its ease of application. However, the optimization steps turned out to be quite critical, especially for sequences with high melting temperatures embedded in lower melting temperature fragments. Considering only the common sequences analyzed by the two techniques, four of the 46 positive samples detected by DGGE were not revealed by DHPLC. This result stresses the limited sensitivity of DHPLC compared with DGGE under the conditions described in this study.

Flow cytometry and quantitative immunohistochemical study of cell cycle regulation proteins in invasive breast carcinoma: prognostic significance.

BACKGROUND: Between January 11, 1991 and January 8, 1992, 104 patients with previously untreated, invasive, primitive breast carcinoma were admitted to the authors' hospital. METHODS: For each patient, flow cytometry DNA analyses on frozen samples and on immunohistochemical staining were performed, including Ki-67, cyclin A, p53, and p21(waf1) (p21), with assessment of the percentages of positive nuclei were assessed. Correlations with classic clinicopathologic data and survival (overall, metastasis free, or recurrence free) and a multivariate analysis were performed. RESULTS: After a multivariate analysis according to a Cox model that was stratified by age, tumor size, tumor grade, lymph node status, and receptor status, among the factors studied, the presence of p21 was the unique remaining prognostic factor for patients with invasive breast carcinoma. Because of the lack of a correlation between p21 and proliferative factors (Ki-67, S-phase, and cyclin A), the authors combined p21 with those markers and found that, for the different combinations, after statistical analysis, only p21 combined with S-phase or with cyclin A and lymph node status were salient survival prognostic factors. CONCLUSIONS: Immunohistochemical study of proteins involved in the cell cycle and assessment of proliferative activity using flow cytometric DNA analysis aided the authors in singling out correlations of cyclin A and S-phase, along with p21, with metastasis free survival and overall survival in patients with invasive breast carcinoma. These promising results will require confirmation in a larger series of patients.

[Primary thyroid lymphomas: clinicopathologic study of 30 cases and review of the literature]. / Lymphomes thyroïdiens primitifs: étude clinico-pathologique de 30 cas et revue de la littérature.

During a both retrospective and prospective study of thyroid cancers treated in the Basse Normandie between 1960 and 1999, we have identified 32 patients with thyroid lymphoma. The correct diagnosis was made initially in 69% of all cases. In the other cases, the diagnosis was secondarily corrected after review of the pathological material. According to the REAL classification, 7 (21%) corresponded to low grade MALT lymphomas, 2 to low grade lymphomas, 10 to high grade MALT Lymphomas and 10 (31%) to high grade lymphomas, one plasmocytoma and two unclassified lymphomas. According to the Ann Arbor classification, stage was IE for 56%, IIE for 19%, IIIE for 3% and 9% for IV. Median survival was 28 months with a mean at 61 months. 20 patients died (62%), 12 from the lymphoma and 8 from intercurrent causes. The overall survival at 5 years was 36% (9 5% CI 16 54%). A comparison of our results with those of the literature was performed.