Evaluation of proliferation parameters in in vivo bromodeoxyuridine labelled lung cancers

In a series of 44 bronchial biopsies from patients suspected of having endobronchial lung carcinoma, the validity of proliferating cell nuclear antigen (PCNA) and Ki67 antigen as proliferative indicators was evaluated in ethanol fixed, paraffin embedded tissue. The percentages of cells positive for these markers were compared to the in vivo bromodeoxyuridine (BrdU) labelling index. A good correlation was found between PCNA immunoreactivity and BrdU labelling index, while Ki67-antigen expression showed a significant relation with BrdU labelling index and with PCNA expression. All three parameters showed a trend towards similar values for the individual cases. Based on the fact that Ki67 antigen is expressed in all cycling cells, whereas replicon-associated PCNA and BrdU only reflect the S-phase fraction, the differences between Ki67-antigen scores on the one hand and BrdU and PCNA scores on the other were smaller than expected. In order to determine the degree of concordance between immunohistochemically and flow cytometrically detected proliferation variables, BrdU incorporation was measured using both methods in duplicate bronchial specimens. Discrepancies in labelling indices were observed predominantly in DNA diploid samples, with consistently lower values in the flow cytometrically analysed specimens. In tumour specimens with an aneuploid DNA content, flow cytometric determination of proliferative activity yielded results similar to those obtained by tissue section examination. We conclude that the scores for PCNA and Ki67 antigen, immunohistochemically detected in ethanol fixed, paraffin embedded tissue reflect functional proliferative activity.     

Enhanced polymer one-step staining (EPOS) for proliferating cell nuclear antigen (PCNA) and Ki-67 antigen: Application to intra-operative frozen diagnosis

Enhanced polymer one-step staining (EPOS) is a novel, highly sensitive one-step immunostaining method. This simple and rapid technlque was applied to intra-operattve frozen diagnosis. The markers of choice were proliferating cell nuclear anmen (PCNA) and Ki-67 antigen. These cell prollferation markers were both identifiable in fresh frozen see tions of the human tonsil In approximately 7 min. The suitable staining sequences are as follows. Frozen sections prepared using 3-aminopropyitimethoxysilane-cpated glass slides are immediately fixed, without air drying, for 15s in a mixture of 50% formalin and 50% methanol for PCNA, and in 10% formalln for Ki-67 antigen. After a brief rinse in phosphate-buffered saline (PSS), sections are incubated with the EPOS antibody for 3 min, followed by PBS rinse for 1 min. The peroxidase activity is visualized in diaminobenzidine-H₂O₂ solution containing 10mmol/L imidazole for 2 min. After a light rinse in tap water, the nuclei are briefly counterstained with 5% methyl green. When necessary, endogenous peroxi-dase blockage in 1% periodic acid solution for 1 min is added before the EPOS antibody incubation. This procedure is applicable to frozen sections of gastric cancers, malignant lymphomas, and brain, liver and peritoneal lesions in which differential diagnosis between benignancy and malignancy was required.     

p53 and Ki-67 immunoreactivity and nuclear morphometry of 'carcinoma-in-adenoma' and adenoma of the gall-bladder

Twenty lntramucosal tumors of ‘carclnomaln-adenoma’ and 43 ademas (39 pylorlc gland type, 4 Intestinal type) of the gall-bladder were studied to establish more precise histo-logical criteria of carcinoma or adenoma In cases of ‘carcinoma In pyforic gland type adenoma’, to compare carcinoma In adenoma with pure, that Is, without adenomatous components, carcinoma, and to confirm the benign nature of spin-dle cell fd in the adenomas. Ki-67 and p53 immunostaining and nuclear morphomety were used. Eight pure intramucessl cancers were used as controls. The formalin-fixed, paraffln+mbedded sections were stained with p53 and Ki-67 antibodies. Splndle cell foci were observed only In the adenoma area of the pyloric gland type, wlth a frequency of 23% In 39 adenomas, and of 45% in 20 tumors of carclnoma-lrradenoma. Ki-67 staining was negative in 129 of 130 spin-die cell foci examlned, regardless of their size, and positive in only one focus (550 pm in size, Ki-67 Index 0.2%). All of the spindle cell foci were negative for p53 stain. The Ki-67 positive index was 36.6 ± 5.6% In the 8 pure carcinomas, and 12.5 ± 1.9% in the cancer areas of 16 tumors with carcinoma-in-adenoma, while it was 7.9 ± 1.7% in the adenoma areas of 16 tumors with carcinoma-in-adenoma and 4.9 ± 0.5% in the 32 pure pyloric gland adenomas. The p53-protein over-expression was found in seven of eight pure intramucosal cancers, and in one of 16 cancer components of carclnoma-in-adenoma. However, it was not found in any of 16 adenoma components of carcinoma-in-adenoma, and 35 adenomas. Cells of the cancer tissue of carcinoma-In-adenoma showed a significantly larger nuclear area and a larger nuclear minor axis than those of the pyloric gland type adenomas, as well as other architectural and cytologic abnormalities differing from the features of adenomas. These results suggest that clustered spindle cells do not indmte a malignant transformation of adenoma cells and that carcinomas in carcinoma-in-adenoma are dtfferent from pylorlc gland type adenomas In terms of morphology and proliferative activity. Moreover, the results of the present study indicate that carcinomas In carcinoma-ln-adenoma are lower In malignancy than pure carcinomas, and that their genetic abnormaltty may differ from that of pure carcinomas.     

Laser microdissection and pressure catapulting (LMPC) in paraffin sections mounted on glass slides. A methodological report

The technique of laser microdissection together with laser pressure catapulting (LMPC) is demonstrated in paraffin sections obtained from surgical specimens of brain tumors mounted on glass slides. A sufficient and precise application of microdissection techniques in tissue on glass slides is worthwhile, since it offers the possibility of a retrospective analysis of archived paraffin sections in histopathology. We could demonstrate a precise dissection of areas in tissues of different thicknesses (4 microm and 20 microm). Areas of tissue mounted directly on glass need to be dissected in a scanning mode in order to remove the total region in form of small tissue fragments row by row. This mode provided a precise microdissection of tissue areas of different sizes and shapes. A successful molecular biological analysis of the microdissected regions could be demonstrated. As an example for such an analysis, differential-PCR for detecting an amplification of the gene for the epidermal growth factor receptor (EGFR) was performed.     

Ultrastructural study of 4 cases of Ki-1 positive large anaplastic cell malignant lymphoma

Summary The ultrastructural morphology of 4 cases of large anaplastic cell malignant lymphoma (Ana ML) is reported. Three cases were primary Ana ML and one pleomorphic large T cell lymphoma with some Ki-1 positive cells. All were confirmed by immunohistochemistry on frozen and paraffin sections. The Ki-1 and EMA positive tumour cells had an abundant cytoplasm, with no differentiation and large pale nuclei with multiple compact or dispersed nucleoli. The morphology is that of an activated cell engaged in protein synthesis and/or in the mitotic cycle. These tumour cells resemble to the Hodgkin's and monolobated Reed-Sternberg cells described in Hodgkin's disease.     

The anaplastic variant of centrocytic lymphoma is marked by frequent rearrangements of the bcl-1 gene and high proliferation indices

Ten cases of classic centrocytic lymphoma as defined in the Kiel classification system were investigated for their immunophenotype, their proliferation activity and by means of molecular diagnostics. The findings were compared to those obtained from a group of nine cases of anaplastic centrocytic lymphoma. Both groups showed virtually identical immunohistochemical characteristics with positivity for CD5 and negativity for CD10 and CD23. In the group of anaplastic centrocytic lymphoma, there were considerably higher proliferation indices as documented by staining for the Ki-67 antigen, up to 80% of the tumour cells being positive. Moreover, the cases of anaplastic centrocytic lymphoma had bcl-1 gene rearrangements in eight out of nine cases compared with three out of 10 cases of classic centrocytic lymphoma. DNA analysis was not able to detect bcl-2 gene rearrangement in any case, pointing to a difference compared with lymphomas of germinal centre origin. The coincidence of anaplastic and sometimes blast-like morphology of the tumour cells, high proliferation index and a rearranged bcl-1 gene in nearly all cases of anaplastic centrocytic lymphoma support their classification as high-grade malignant variants of centrocytic lymphoma and suggest a possible role for the bcl-1 locus not only in the origin but also in the progression of centrocytic lymphomas.     

Telomerase activity and proliferation index in aggressive mature B-cell lymphoma: comparison to germinal center phenotypic markers

Cell proliferation may be evaluated by various methods, including Ki-67 immunohistochemistry and measures of telomerase activity. Both methods would theoretically show comparable increases in a given case. To evaluate the relationship between these 2 markers of proliferation in aggressive mature B-cell lymphomas, 48 cases were studied. The study group included 5 cases of mantle cell lymphoma (MCL); 6 cases of Burkitt's/Burkitt's-like lymphoma (BL); 9 cases of follicular lymphoma, grade 3 (FLC); and 28 cases of diffuse large B-cell lymphoma (DLC). Telomerase activity was measured as total product generated (TPG) units, and TPG results for the aforementioned cases were compared to the TPG results for 10 cases of reactive follicular hyperplasia. An overlap in TPG scores between reactive cases and lymphoma cases was found. Significant differences in both log TPG (P = 0.0443) and Ki-67 (P = 0.0006) were seen in the different lymphoma types. A positive correlation between Ki-67 percentage and TPG score was identified in FLC (r = 0.9281; P = 0.0003), but a poor correlation between these 2 indicators was seen in the other lymphoma types. Cluster analysis identified distinct patterns for MCL, FLC, and BL, but heterogeneous patterns for DLC. Because increases in both Ki-67 proliferation and telomerase activity are reported in normal germinal centers (GCs), these tests were also evaluated for usefulness as markers of a GC cell phenotype. Among the FLC and DLC cases, features of a GC phenotype significantly correlated with increased Ki-67 percentage (P = 0.0152), but not with increased log TPG. An elevated log TPG correlated with CD10 expression, and elevated Ki-67 percentage correlated with both CD10 and BCL-6 expression. TPG level and Ki-67 percentage did not correlate with the presence of t(14;18) or BCL-2 protein expression. Although the proliferation patterns were fairly distinctive for MCL, FLC, and BL, these studies show that markers of cell proliferation do not by themselves,identify distinct subtypes of large cell lymphomas. With the exception of FLC, the tumors exhibited poor correlation between telomerase activity and Ki-67 proliferation index. These tests did show some correlation with expression of GC cell phenotypic markers, however.     

Proliferative activity in benign and neoplastic prostatic epithelium

The increasing availability of means for the early detection of prostatic cancer has brought under scrutiny the criteria used for prognosis and emphasized our limitations in understanding what determines the rate of progression in these cancers. The rate of cancer cell proliferation has been under intense investigation, which, however, has yielded conflicting results. In this study we evaluated the proliferative activity of benign and neoplastic prostatic epithelium, using various existing methodologies. We first analysed the variability introduced by the methodological approach and then attempted to demonstrate whether determination of the proliferative capacity had any clinical consequence that complemented the histological grading. Tissue samples from patients, 88 with cancer and 46 with benign prostatic pathology, were studied using in vitro bromodeoxyuridine (BrdU) incorporation as well as Ki67 and the proliferating cell nuclear antigen (PCNA) to estimate the proliferative activity. Increased proliferation was found consistently in inflammation and metaplasia, but not in hyperplasia. In contrast, cancers showed marked variability. Although average proliferation indices increased with grade, there was a wide scatter of values. Correlation was stronger with stage, but also depended on the methodology. Bromodeoxyuridine indices over 10 per mille had a positive predictive value of 79 per cent for cancers extending beyond the prostatic capsule and may prove particularly helpful for evaluating patients with grade 7 cancer. This observation is significant, since grade 7 cancers are the most frequent and the least predictable.     

Replicative MCM7 protein as a proliferation marker in endometrial carcinoma: a tissue microarray and clinicopathological analysis

AIMS: To assess, in tissue microarray (TMA), the proliferative activity of endometrial carcinoma using one of the minichromosome maintenance (MCM) proteins (MCM7), and to explore its potential value for prognosis. MCM proteins are essential for eukaryotic DNA replication and have recently been used to define the proliferative compartments in human tissues. METHODS AND RESULTS: Immunohistochemistry for MCM7 and Ki67 was performed on TMAs constructed from 212 cases of endometrial carcinoma. MCM7 and Ki67 expression was quantified according to the extent of nuclear staining. An analysis was carried out of the association between MCM7 expression and that of Ki67 and the clinicopathological characteristics of endometrial carcinoma. MCM7 and Ki67 immunoreactivity was clearly evident in the nuclei of tumour cells. MCM7 and Ki67 labelling indices in endometrial carcinomas correlated with each other (P < 0.001). A significant correlation existed between the MCM7 labelling index and histological grade (P = 0.008) and patients' age at diagnosis (P < 0.001). Well-differentiated carcinomas and younger patients had a lower MCM7 index. Poor survival was observed in patients with endometrial carcinoma with a high MCM7 index (P = 0.03) and MCM7 was found to be an independent prognostic factor by multivariate analysis (P = 0.04). The Ki67 labelling index correlated with histological grade (P = 0.01) but had no significant prognostic impact (P = 0.50). CONCLUSIONS: In this TMA study on endometrial carcinoma, MCM7 was found to be a more reliable and useful marker than Ki67 in assessing tumour proliferation and in the prognosis of patients.