Comparison of liquid based cytology and histology for the evaluation of HER-2 status using immunostaining and CISH in breast carcinoma.

BACKGROUND: HER-2 amplification is an important prognostic biomarker and treatment determinant in breast carcinoma. AIMS: To correlate immunocytochemical (ICC) expression of HER-2 and gene amplification determined by chromogenic in situ hybridisation (CISH) using liquid based cytology (LBC) with immunohistochemistry (IHC) and CISH using histological samples of the same breast carcinomas. METHODS: Frozen sections and cytobrushings of 103 breast carcinomas were analysed. Four techniques were performed on each tumour: two on LBC samples (ICC, and CISH, both graded as positive, indeterminate, or negative) and two on histological samples (IHC and CISH). Two cell lines (MCF-7, negative; BT 474, positive) were used as controls for cytological analysis. A complementary fluorescence in situ hybridisation technique was carried out in histological samples with low amplification (4-10 dots/nucleus). RESULTS: Interobserver agreement for the four techniques calculated by the kappa coefficient indicated a substantial agreement. Nine cases failed in cytology because of poor cellularity. Among 94 cases, 19 were amplified; 73, 12, and 9 tumours were scored 0 or 1+, 2+, and 3+, respectively by IHC and 75, 13, and 6, respectively, by ICC. CISH found no amplification in 72 tumours. Correlations between the IHC and CISH results in the histological and cytological samples were always significant. CONCLUSIONS: Her-2 status could be determined in LBC samples and correlated well with reference histological methods using in situ hybridisation. ICC was less reliable because of the presence of the cytoplasmic membrane. However, these results should be confirmed by a large multicentre study.

Less HLA-G expression in Plasmodium falciparum-infected third trimester placentas is associated with more natural killer cells.

During pregnancy, maternal immune tolerance of the fetal semi-allogeneic graft is partly the consequence of extravillous trophoblast HLA-G expression and its interaction with natural killer (NK) cells. Plasmodium falciparum malaria is frequently associated with maternal and fetal complications. Local HLA-G expression and the number of NK cells were evaluated immunohistochemically in P. falciparum-infected and uninfected placentas (15 each) collected in a seasonal malaria-hypoendemic area. In control placentas, HLA-G was almost always expressed in extravillous trophoblast whereas, in infected placentas, it was significantly more weakly expressed in extravillous trophoblast but was also detected in intervillous space macrophages. NK cells were evaluated in intervillous and intravillous spaces and in basal plate. NK cells were always more abundant in basal plate than in intervillous and intravillous spaces in infected or control placentas. For each area, more NK cells were seen in infected than control placentas. These data suggest that HLA-G down-regulation and more NK cells in placentas may be among the mechanisms involved in poor birth outcome associated with P. falciparum infection.

Proliferation assessment in breast cancer: a double-staining technique for AgNOR quantification in MIB-1 positive cells especially adapted for image cytometry.

There are two ways of measuring the cell proliferation. The first one consists of quantifying the number of cycling cells with the help of antibodies directed against cells either in G1, S, G2 or M phase. The second way is to assess the cell cycle duration by the quantification of AgNOR proteins. Measuring both the features on the same slide represents an attractive way to tackle the proliferating activity of a cell culture or a tumor. Here, we propose a MIB-1 and AgNOR double staining method especially adapted to image cytometry measurement using MIB-1 antibody coupled to FITC in order to avoid the thresholding problems encountered with such a multilabeling technique. We have applied this new method on a series of 39 breast cancer cases, with at least 4 years follow-up, in order to determine the prognosis significance of this measurement. MIB-1 alone is not linked to prognosis, while the global mean AgNOR area is significantly linked to prognosis in terms of development of visceral metastasis or death. However, the global mean AgNOR area is insufficient to determine the time limit of appearance of metastasis or relapse. Our results clearly demonstrate that a high mean AgNOR area within a cell population having a high MIB-1 index can discern tumors with a high metastatic potential. By multiplying AgNOR area by the percentage of MIB-1 positive cells we calculate the proliferative activity, P, which brings very important information concerning the time limit of relapse.

Semiautomatic quantification of silver-stained nucleolar organizer regions in tissue sections and cellular smears.

Silver staining of nucleoli reveals argyrophilic proteins associated with nucleolar organizer region (Ag-NOR) proteins. Argyrophilic components appear as dots about 1 micron in diameter dispersed throughout the nucleolus (Ag-NOR dots). The count of Ag-NOR dots is a useful index for improving the cancer diagnosis and determination of prognosis. Here we describe software developed on a medium-cost image analyzer in order to evaluate the mean area of NORs and their number relative to an internal reference, the number and areas of clusters of NORs and the area of the nucleus. Statistical analysis of the data was performed during counting. The first application concerned counting NOR dots during mitosis in cell imprints; those counts were 2.3, 15.3 and 55.56 for the metaphase, telophase and interphase, respectively (relative to unitary dots of metaphase cells). In the second application we demonstrated a significant difference in NOR numbers between two groups of prostatic cancers with good and poor prognoses (6.05 +/- 2.79 SD and 7.96 +/- 3.01, respectively; with Student's t test, = 1.999; P = .05).

[Silver staining of nucleolus organizer regions (NORs). Application to the study of nucleolar structure and value in pathology]. / Coloration des organisateurs nucléoraires (NORs) par l'argent. Application à l'étude de la structure du nucléole et intérêts en pathologie.

Nucleolus is the morphologic expression of synthesis and maturation of ribosomal RNA (rRNA) from amplified ribosomal DNA (rDNA). Nucleolar Organizer Regions (NORs) are functional subunits of the nucleolus in which actively transcribed rDNA is surrounded with numerous regulatory proteins. Some of them are argyrophilic non-histone proteins (Ag-NOR proteins). By using a cytochemical reaction based on this argyrophilia, active NORs may be stained by the precipitation, at their level, of metallic silver granules whose quantity is directly related to the nucleolar activity. In the present paper, we described various applications of a silver-staining method we developed in our laboratory. The Ag-NOR proteins were ultrastructurally localized within precise nucleolar components. Moreover by viewing thick-sections of silver-stained cells with high-voltage microscopes we were able to describe the three-dimensional structure of nucleoli. Finally, this silver-staining method may be applied at the optical level, to sections of routinely fixed and paraffin-embedded human tissues. With this simple staining method, it is now possible to study the relationships of the number of nucleolar argyrophilic structures with the diagnostic of neoplasms.