Estimation of the proliferative activity of human breast cancer tissue by means of the Ki-67 and MIB-1 antibodies--comparative studies on frozen and paraffin sections

The estimation of the Ki-67 index in human breast cancer tissue has been proven to be a useful prognostic tool. The examination can be performed, however, only on frozen sections (FS). The development of an antibody directed against parts of the Ki-67 antigen (MIB-1) has opened a new route to determine the proliferative activity on paraffin sections (PS). MIB-1 immunohistochemistry is used instead of Ki-67 immunohistochemistry if a tumour is delivered to the pathologist after formalin fixation or if that part of the tissue suspicious for breast cancer must be totally embedded in order to confirm the diagnosis. The present study compares the findings of Ki-67 (FS) and MIB-1 (FS and PS) immunohistochemistry in a total of 544 cases of human breast cancer. The findings confirm a good statistical correlation between the Ki-67 and the MIB-1 findings. The MIB-1 results are 2-2.5 times higher in FS than in PS. Good agreement exists between the Ki-67 indices determined on FS and the MIB-1 indices determined on PS. If the cut-off value for the separation of Ki-67 negative and positive cases is defined as 10%-20%, a MIB-1 index in PS of 10% permits the correct prediction of a negative Ki-67 index in 97% of the cases, and a MIB-1 index of 30% or more correctly predicts a positive Ki-67 index in 90% or more of the cases. Hence, the determination of the MIB-1 index on PS may replace the determination of the Ki-67 index on FS with a high degree of probability.     

The correlation of Ki-67 staining indices with tumour doubling times in regrowing non-functioning pituitary adenomas

In order to improve our ability to predict the regrowth of nonfunctioning pituitary adenomas, we tried to assess the correlation between growth fractions with Ki-67 and PCNA (proliferating cell nuclear antigen) and tumour doubling times in regrowing tumours, and also to find out any difference of growth fractions between the regrowing and the cured cases. In 33 patients with non-functioning pituitary adenomas, 14 cases including 11 with cavernous sinus invasion showed residual tumour on MRI after the operation (regrowing group) and 19 cases had no tumour regrowth on MRI within 5 years after the operation (cured group). Immunocytochemical studies were done with monoclonal antibodies (anti-PCNA, anti-Ki-67: MIB-1). The growth fraction of each tumour was estimated by calculating the ratio of the positive nuclei to the total number of tumour cells with the aid of an image analyser (Mac SCOPE). The tumour doubling times were estimated from serial CT or MRI with the aid of the image analyser (NIH image). Ki-67 staining indices ranged from 0.2% to 1.5% (n = 14, 0.86 +/- 0.10%; mean +/- SEM) in the regrowing group, and from 0.1% to 0.5% (n = 19, 0.23 +/- 0.03%) in the cured group. PCNA staining indices of the regrowing group ranged from 0.6% to 24% (n = 14, 3.7 +/- 1.6%). In the regrowing group, the tumour doubling times ranged from 200 to 2550 days (930 +/- 180 days), and showed a significant inverse correlation with Ki-67 staining indices, but no correlation with PCNA staining indices. The regrowing group showed a significantly higher Ki-67 staining index (n = 14, 0.86 +/- 0.10%) than the cured group (n = 19, 0.23 +/- 0.03%) (p < 0.01). These results indicate that immunocytochemical studies using MIB-1 may be better than those with PCNA for the prediction of regrowth in non-functioning pituitary adenomas. Immunocytochemical study with MIB-1 could lead to the accurate prediction of the rapid regrowing lesions in non-functioning adenomas.     

Arrhythmogenic right ventricular dysplasia/cardiomyopathy: review for the clinician

The objectives of this study were to measure the proliferation indices in canine mammary tumors using immunohistochemical detection of Ki-67 and proliferating cell nuclear antigen (PCNA), to determine the relationship of these antigens to clinical and pathologic variables, and to investigate the usefulness of these antigens as prognostic indicators. Ninety-six female dogs with 115 primary nonmetastasized spontaneous mammary tumors and dysplasias were included in the study. Immunostaining was performed using MIB-1 and PC10 monoclonal antibodies against Ki-67 and PCNA, respectively. Ki-67 and PCNA proliferation indices were determined. Dogs were followed for 18 months, with clinical examinations every 3-4 months. There was a significant correlation between Ki-67 and PCNA indices in the dogs with dysplasias and benign tumors but not in the dogs with malignant tumors. The clinical stage at first presentation was related to the proliferative index measured with Ki-67 but not to that measured with PCNA. Proliferation indices were significantly lower in the nonmalignant tumors and dysplasias than in the malignant tumors. In malignant tumors, the PCNA index had a positive correlation with the histologic malignant grade and the nuclear grade. High index values of Ki-67 were positively correlated with metastasis, death from neoplasia, low disease-free survival rates, and low overall survival rates. PCNA displayed no significant association with these variables. Multivariate analyses concerning metastasis, disease-free survival, and overall survival revealed that the Ki-67 index had prognostic value.     

Morphologic, immunophenotypic, and molecular evaluation of bone marrow involvement in non-Hodgkin''s lymphoma

AIM: To determine the tumour proliferative activity in a series of archival cerebral astrocytomas and compare proliferating cell nuclear antigen (PCNA) and Ki-67 labelling indices in the primary and recurring neoplasms following therapeutic radiation. METHOD: Twenty eight cases of pre-irradiated and post-irradiated astrocytomas (ranging from WHO grades I to IV) were stained immunohistochemically using the avidin-biotin horseradish peroxidase technique. Two antibodies, PC10 and MIB-1, were used to establish the proliferating labelling indices, PC10 identifies PCNA and MIB-1 recognises the Ki-67 antigen. RESULTS: Both antibodies showed significantly higher labelling indices in the post-irradiated specimens. However, in general, the Ki-67 indices were lower than those for PCNA. MIB-1 immunoreactivity showed less variation and was more intense than that seen with PC10. The discrepancy between the labelling indices of the pre-irradiated and post-irradiated samples raises questions about the evolution of astrocytomas and the effects of therapeutic intervention. CONCLUSIONS: The data may represent genetic alterations, the natural tumour course, and/or the effect of radiation. Although both of the antibodies reflected the state of growth of neoplastic cells in astrocytomas, MIB-1 was more reliable. A simple immunohistochemical method using proliferation markers does have an important role in the future care of patients with astrocytoma.     

Evaluation of Ki67 antigen using MIB1 antibody as a prognostic factor in renal pelvic and ureteral cancer

(PURPOSE). Recently, several reports showed that immunohistochemistry using MIB-1 antibody, which recognizes Ki-67 antigen, is one of the useful methods to determine the proliferative activity in various cancer. To evaluate the prognostic usefulness of the MIB-1, antibody we assessed the cell proliferation immunohistochemically in urothelial cancer. (METHODS). The proliferative activity of thirty cases of renal pelvic and ureteral cancer has been investigated immunohistochemically using MIB-1 antibody, which recognizes Ki-67 antigen, a human nuclear antigen expressed in proliferating cells. (RESULTS). The Ki-67 index correlated with prognostic factors such as pathological stage and histological grade. The patients with early stage or low grade tumors had lower Ki-67 indices. And the Ki-67 index significantly correlated with recurrence and prognosis. The tumors of patients with recurrence or cancer death had higher Ki-67 indices. When the patients were suggrouped according to Ki-67 indices (more than 22%) had significantly worse prognosis even in the same grade. Especially in the grade 2 group, all the four patients in the higher Ki-67 index subgroup had recurred and died of cancer, whereas, in the subgrouped patients with tumors of lower Ki-67 indices, only three patients had bladder cancer recurrence, and no patients had died of cancer, except one case with advanced tumor (T4, N3, M0) resected incompletely. (CONCLUSIONS). These results indicate that the Ki-67 index is a useful prognostic factor and may enhance the prognostic accuracy determined by conventional morphological grading systems.     

Evidence for the continuity of early problem behaviors: application of a developmental model

Mitotic index > 6, proliferating cell nuclear antigen (PCNA) index > 5%, high tumour grade and absence of progesterone receptors (PR) are significant predictors for poor outcome in meningiomas. Since MIB-1 (Ki-67) is a more specific cell proliferation marker, and overexpression of TGF-alpha is also associated with tumour progression, we compared the prognostic significance of these factors with the other indices. Intracranial meningiomas from 21 men and 36 women (age 54.5 +/- 1.7, mean +/- SEM) were classified as 24 benign, 24 atypical and nine malignant. Twenty-one of the 57 tumours recurred (mean interval to recurrence was 57.3 +/- 13.1 months). The mean follow-up period for patients without tumour recurrence was 81.9 +/- 8.7 months. MIB-1 labelling index (LI) was expressed as percentage of labelled nuclei to total tumour nuclei counted in the most densely labelled areas. Analysis of variance revealed significant differences between tumour grades for MIB-1 labelling indices (0.75 +/- 0.21 for benign, 3.2 +/- 0.57 for atypical 6.04 +/- 1.48 for malignant; P < or = 0.0066), and between malignant and non-malignant meningiomas for TGF alpha staining scores (P < or = 0.029). MIB-1 LI also correlated with mitotic and PCNA indices (P < or = 0.0001), but not with age of the patients. Male patients had higher tumour MIB-1 LI than females (P < or = 0.0128). Univariate analysis indicated that MIB-1 LI > 3%, TGF alpha score > 4 (scoring scale 0-5), mitotic index > 6, and negative PR status were significant factors for worse outcome. Higher MIB-1 LI, TGF alpha score and mitotic index as continuous variables were also significant negative predictors. With multivariate analysis, both MIB-1 LI and TGF alpha score remained significant factors when paired with all other variables: TGF alpha or MIB-1 LI, respectively, mitosis, PCNA, tumour grade, PR status, age, sex, postoperative radiation therapy. We conclude that MIB-1 LI and TGF alpha score are important independent prognostic indicators for patients with meningiomas.     

Quantitative analysis of cellular proliferative activity in 35 T-cell non-Hodgkin's lymphomas. Use of proliferating cell nuclear antigen and Ki-67 (MIB-1) antibodies and nucleolar organizer regions

OBJECTIVE: To analyze-cellular proliferation in non-Hodgkin's lymphoma (NHL) of T-cell origin using three variables available on histologic paraffin, dewaxed sections. STUDY DESIGN: The study group consisted of 35 T NHLs (22 low and 13 high grade). Two immunohistochemical methods established the percentage of cells expressing proliferating cellular nuclear antigen (PCNA), or PCNA index, and the Ki-67 antigen, or MIB-1 index. The third method quantified nucleolar organizer regions (NORs) with an image analyzer, giving the NOR area and number of NORs; an internal reference on lymphocytes was used. RESULTS: For the PCNA index, each subtype of low grade NHL demonstrated a difference as compared with high grade NHL except for angioimmunoblastic type NHL (AILD). The difference between the two grades became significant after including AILD-type NHL within high grade NHL (P = .02). The MIB-1 index gave similar results. The PCNA index and MIB-1 correlated (r = .55, P = .008). The relative NOR area and number of NORs differed significantly between the two grades (P < 10(-4) and P < 10(-2); the absolute NOR area differed to a lesser degree (P = .02), and no difference was observed for the absolute number of NORs (P = .07), stressing the importance of an internal reference. NOR areas and numbers correlated highly (r = .90 for relative and .78 for absolute variables, P < 10(-4)). No relation was found between PCNA and MIB-1 indexes. CONCLUSION: Correlations between these variables and grades of malignancy, between the two indices with each other and between the AgNOR variables with each other, including referring to internal lymphocytes, were reported for these T NHL-like tumors in studies on B NHL. The proliferative character of the AILD-type T NHL was in accordance with their worse prognosis. The absence of a correlation between PCNA or MIB-1 indices and NOR variables may reflect a biologic difference between B and T NHLs in a shorter cell cycle or more important functional activity in T NHL.     

Quantitative three dimensional echocardiography in patients with pulmonary hypertension and compressed left ventricles: comparison with cross sectional echocardiography and magnetic resonance imaging

Meningiomas from 40 adult patients were labeled immunohistochemically with monoclonal antibodies to bromodeoxyuridine (BUdR) and the Ki-67 antigen, MIB-1. The meningiomas were classified as classical, or benign (n = 31); atypical (n = 4); or malignant (n = 5). Meningeal sarcomas and hemangiopericytomas were excluded. The patient population consisted of 26 women and 14 men, ranging in age from 26 to 75 years. BUdR proliferation indices ranged from 0% to 5.8%, measurements that were expectedly lower than those for MIB-1, which ranged from 1.5% to 19.3%. MIB-1 proliferation indices were not significantly affected regarding steroid pretreatment or age. These results show a good correlation between the BUdR and MIB-1 proliferation markers (rs = 0.72; P < .0001), which supports the use of anti-MIB-1 as an alternative labeling tool to BUdR for the determination of the proliferation index in meningiomas, thus avoiding the administration of a potentially mutagenic drug.     

A phase II trial of weekly infusional 5-fluorouracil in combination with low-dose leucovorin in patients with advanced colorectal cancer

We examined 59 breast cancers for p53 and bcl-2 protein expression by immunohistochemistry. The results were correlated with Ki-67 immunostaining. p53-negativity was noted in 40 cases and the remaining 19 tumours were p53-positive. Thirty-six tumours showed strong expression of bcl-2 and in 23 no staining for this protein was observed. We found statistically significant reverse correlation between expression of p53 and bcl-2 in majority of carcinomas: 31 cases were bcl-2 positive and p53-negative, and 14 tumours were bcl-2-negative and p53-positive. Six carcinomas showed no nuclear staining for Ki-67 and in the remaining 53 the percent of cancer cells positive for Ki-67 ranged from 1 to 60 (mean: 14.6). In these 53 cases we found that bcl-2-positive tumours were characterized by lower proliferation than bcl-2-negative tumours, the mean value of Ki-67 immunostaining being 10.7% and 23.0%, respectively. p53-negative tumours showed lower proliferation than p53-positive tumours: mean Ki-67 index was 10.2% and 23.9%, respectively. We conclude that immunohistochemically detected p53 and bcl-2 proteins show a significant inverse relationship in majority of breast carcinomas and their expression correlates with tumour proliferation (Ki-67 immunostaining).     

Ki-67 labeling indices in non-small cell lung cancer: comparison between image cytometry and flow cytometry

Expression of the proliferation-associated nuclear antigen Ki-67 was studied in 27 human non-small cell lung cancers (NSCLCs). We measured immunohistochemical Ki-67 labeling indices using image cytometry (ICM) and flow cytometry (FCM) and compared the indices determined with the two methods. Ki-67 labeling indices of tumor cells ranged from 2% to 59% with ICM, and from 3% to 56% with FCM, varying from case to case. There was a linear correlation of values between the two methods (r = 0.77, P < 0.05). To examine whether Ki-67 labeling indices represent tumor proliferative activity, we studied the relationship between the Ki-67 labeling index and the S-phase fraction determined by FCM. There was a positive correlation between the Ki-67 labeling index and the S-phase fraction (r = 0.91, P < 0.01). Comparison of Ki-67 labeling indices and clinicopathological parameters showed lower Ki-67 labeling indices in well-differentiated tumors than in moderate and poorly differentiated tumors (P < 0.05). No correlation was observed between Ki-67 labeling indices and p53 expression. It was concluded that the Ki-67 labeling index was in fact measured with both ICM and FCM, and that it represents tumor proliferative activity of NSCLCs.     

Aberrant expression of cyclin A and cyclin B1 proteins in oral carcinoma

Cyclins play an important role in regulating the passage of dividing cells through critical checkpoints in the cell cycle. Aberrant expression of cyclin proteins has been found in a number of human cancers, including carcinomas of the head and neck, where amplification of the cyclin D1 gene is a common finding. The objective of this study was to examine cell cycle kinetics in oral carcinomas by determining the expression of the S phase protein cyclin A and the M phase protein cyclin B1. Routinely processed tissue sections of 50 oral squamous cell carcinomas from the floor of the mouth were stained by immunohistochemistry for cyclin A, cyclin B1 and Ki-67 proteins. Ten specimens of normal epithelium from the floor of the mouth were used as controls. The number of cells showing nuclear staining for cyclin A, cyclin B1 and Ki-67 proteins was determined by computer image analysis. There were 17 well-differentiated, 25 moderately differentiated and 8 poorly differentiated tumours. Mean counts for cyclin A (29.50+/-4.10, mean+/-95% CI), cyclin B1 (2.05+/-0.30) and Ki-67 (49.46+/-5.91) proteins in the carcinomas were significantly higher than counts for the normal epithelial controls (cyclin A: 9.30+/-1.72; cyclin B1: 1.01+/-0.36; Ki-67: 17.40+/-4.17). For cyclin A, cyclin B1 and Ki-67, mean staining scores for all tumour grades were significantly higher than controls. There was a strong correlation between Ki-67 and cyclin A scores in all tumour groups (r2=0.68); however, the correlations between cyclin B1 and cyclin A scores (r2=0.35) and between cyclin B1 and Ki-67 scores (r2= 0.39) were weak. We conclude that there is overexpression of cyclin A and cyclin B1 proteins in oral carcinoma. Furthermore, the poor correlations for cyclin B1 scores with other cell cycle indices suggest that there may be aberrant cell cycle progression at the G2/M checkpoint in oral carcinomas.     

Ki-67 and p53 in T2 laryngeal cancer

OBJECTIVE: To study the relationship between the proliferative capacity, represented by the immunohistochemical labeling index (LI) of proliferation marker Ki-67, and the p53 status, as in theory an intact p53 cell cycle checkpoint system should result in a lower proliferative capacity. STUDY DESIGN: From a group of 128 patients with a T2 laryngeal carcinoma, presented from 1989 to 1993 at the University Hospital Utrecht, 20 patients with recurrent disease and 16 patients without recurrent disease were randomly selected. All patients received primary irradiation. METHODS: Denaturing gradient gel electrophoresis and immunohistochemistry determined the p53 status. MIB-1 staining was used to determine the Ki-67 LI. RESULTS: In 36% of specimens we found a p53 mutation with overexpression (LI, 31%). In 8% a p53 mutation without p53 overexpression was found (LI, 18%). Forty-two percent showed no mutation but, nevertheless, overexpression (LI, 35%). Neither mutation nor overexpression was found in 14% (LI, 38%). No correlation exists between p53 status and proliferative capacity of tumors (analysis of variance [ANOVA]; P = .104). The proliferation rate as established with Ki-67 LI positively correlates with response to radiotherapy (P = .006). CONCLUSIONS: 1. Overexpression of wild-type p53 protein does not result in cell cycle arrest measurable by a lower Ki-67 LI in comparison with cases overexpressing mutant type p53 protein. 2. A high Ki-67 LI correlates with a favorable response to radiotherapy.     

Gene expression of steroidogenic enzymes in chicken embryonic gonads

The monoclonal antibody Ki-S2 binds to a recently characterized proliferation-specific protein, p100. To assess its distribution pattern under physiologic and pathologic conditions, we performed immunohistochemical analyses on an exhaustive spectrum of normal tissues, 624 miscellaneous solid cancers, and 95 hematologic malignancies, and compared the results with Ki-67 immunostaining on consecutive sections. In addition, Ki-S2 expression was related to the DNA content by dual parameter flow cytometric analysis in parallel with Ki-67 labeling using a human cancer cell line. Immunoreactivity was enhanced at the G1/S transition and persisted through G2 and M phase. After adequate antigen retrieval, the antibody was found to yield identical results on fresh and formalin-fixed, paraffin-embedded material. The antibody specifically labeled actively proliferating cells, which constitute a subset of the population recognized by Ki-67. In normal human tissues, Ki-S2 immunolabeling hardly ever exceeded 40% of the Ki-67+ cell fraction. Immunoreactive scores of the two antibodies exhibited a linear correlation, but statistically significant differences in the ratio of Ki-S2-positive to Ki-67-positive cells were nevertheless observed between different tissue types. In contrast, the ratio of Ki-S2 and Ki-67 immunoreactive scores varied widely in neoplastic cells and tissues, occasionally attaining a ratio of almost 1:1. This suggests that loss of growth regulatory mechanisms in malignant cells might result in an extreme reduction of the G1 phase fraction and thus in a significantly shorter doubling time. Therefore, antibody Ki-S2 is likely to allow a more precise evaluation of the cell fraction that will complete a division cycle and a more confident appraisal of the malignancy potential of a neoplastic process.     

Prognostic value of Ki-67 compared to S-phase fraction in axillary node-negative breast cancer

The primary purpose of this study was to evaluate the prognostic significance of Ki-67, a cell proliferation-associated antigen, in a large group (n = 674) of axillary node-negative breast cancer cases with long-term follow-up and to correlate Ki-67 antigen expression with S-phase fraction. Ki-67 immunostaining was assessed both semiquantitatively and quantitatively. The statistical analysis focused on agreement between methods of Ki-67 quantification, agreement between Ki-67 and S-phase fraction, associations between Ki-67 and other clinical variables, and prognostic value of Ki-67. There was excellent agreement between the two methods of Ki-67 assessment (Spearman rank correlation, rsp = 0.91; P = 0.0001; n = 674) but only weak correlation between either semiquantitative or quantitative Ki-67 and S-phase fraction (rsp = 0.12 and rsp = 0.15, respectively). Ki-67 (overall median, 2%) was independent of tumor size and modestly related to other measures of tumor aggressiveness. Using a cutpoint of 5% (percentage of tumor cells), cases with high Ki-67 exhibited a significantly shorter disease-free survival (Padj = 0.004). In multivariate analysis, high Ki-67 was associated with a 1.8-fold increased risk of recurrence (P = 0.001). In the subgroup with S-phase data, the adjusted relative risk (hazard ratio, 1.9; P = 0.02) was unchanged by inclusion of S phase in the model. This suggests that Ki-67 provides significant independent prognostic information in addition to that contained in tumor size and S-phase fraction.     

Immunohistochemical assessment of the MIB-1 Labeling Index in human hepatoblastoma and its prognostic relevance

BACKGROUND/PURPOSE: The MIB-1 monoclonal antibody has been raised against recombinant parts of the Ki-67 antigen, which is a cell cycle-related nuclear protein that is elevated in late G1 and S phases. The aim of the study was to analyze the expression pattern of MIB-1 in hepatoblastoma and to assess whether it provides any prognostic information in clinical practice. METHODS: Sections from formalin-fixed paraffin embedded tissues were collected from 18 patients who had hepatoblastoma and stained with MIB-1 antibody according to streptavidinbiotin method. A percentage score of positively stained nuclei, MIB-1 Labeling Index (LI), was determined and correlated with clinical variables. MIB-1 LI ranged from 0% to 39.5% with a mean value of 13.5%. Among them in 14 patients, who received preoperative chemotherapy, the authors analyzed the result of MIB-1 staining. RESULTS: Although there were no significant correlations between MIB-1 LI and age, sex, or histological type, a statistically significant correlation was found between clinical stage and MIB-1 LI. Mean MIB-1 LI was lower in patients with stage I and II than in those with stages III and IV (P < .05). Metastatic lesions showed higher MIB-1 LI than primary lesions, indicating that metastatic tumor cells have an increased rate of cellular proliferation. Kaplan Meier survival curve showed that patients with MIB-1 higher than 10% (n = 3) had a worse survival rate than those with lower than 10% MIB-1 LI (n = 11), whereas there was no significant difference because of the limited number of cases. Because high MIB-1 LI was correlated with clinical stage and poor survival rate, MIB-1 LI may be considered an important prognostic factor in hepatoblastoma. CONCLUSION: Although further studies, including larger series of patients, are required, the authors consider MIB-1 immunostaining an easy method to assess proliferative activity that provides useful prognostic information in hepatoblastoma.     

Mechanism of induction of rat hepatic CYP2B and 3A by the pesticide methoxychlor

The S-phase which assesses tumor proliferation has been considered to be an independent prognostic factor for breast carcinoma. Quantitative analysis of MIB-1 immunoreactivity is a newly recognized method of determining cellular proliferation that offers some advantages over flow cytometry when limited tumor tissue is available. However, it has been controversial whether there is a significant correlation between MIB-1 immunostaining and S-phase in defining proliferation activity in breast cancer. In order to explore the usefulness of MIB-1 as an additional proliferation parameter and a potential prognostic factor for breast cancer, we analyzed 94 cases of invasive ductal carcinoma of the breast by both flow cytometry (for S-phase and DNA ploidy) and quantitative MIB-1 immunohistochemical analysis using formalin-fixed paraffin-embedded tissue. MIB-1 staining was quantitatively analyzed by image analysis and by visual scoring. Forty-six cases were diploid by flow, while the remaining 48 cases were aneuploid tumors. T-test results indicated that S-phase means were significantly greater (p = 0.0001) in aneuploid cases (mean = 18) compared to diploid cases (mean = 7). MIB-1 means were also greater in aneuploid patients, but these differences were only marginally significant (p = 0.05). S-phase was positively correlated with MIB-1 (r = 0.36, p = 0.003 for image analysis and r = 0.34, p = 0.001 for visual scoring). ROC curve analysis indicated that MIB-1 quantitation is a good predictor of high S-phase (i.e., > 10 percent) in aneuploid cases. A MIB-1 cutoff value of 25 percent for image analysis achieved 82 percent specificity and 80 percent sensitivity for aneuploid high S-phase, while a MIB-1 cutoff value of 40 percent for visual scoring was 73 percent specific and 85 percent sensitive. However, in diploid cases, no comparable MIB-1 cutoffs could be achieved for detecting high S-phase. In summary, our study demonstrated that aneuploid breast carcinomas proliferate more aggressively than diploid tumors. Although linear correlation between MIB-1 and S-phase was weak, MIB-1 was considered to be a good predictor of high S-phase in aneuploid breast cancer patients, possibly due to a threshold effect. Image analysis and visual scoring of MIB-1 immunoreactivity appeared to be comparable in analyzing proliferative activity in breast cancer. Thus MIB-1 assessed by visual scoring may be a less expensive alternative to image analysis.     

Analysis of p53 gene mutation by polymerase chain reaction-single strand conformation polymorphism provides independent prognostic information in node-negative breast cancer

Controversy continues regarding the prognostic utility of detection of p53 gene abnormalities in node-negative breast cancer. To resolve this, we used a rapid and nonisotopic PCR-single strand conformation polymorphism method to screen for mutations in exons 4-8 of the p53 gene in primary tumors from 422 node-negative breast cancer patients. The prevalence of p53 mutation in the exons tested was 18%. p53 mutation was significantly associated with several markers of poor prognosis including larger tumor size, high tumor grade, low hormone receptor content, increased expression of MIB-1 (Ki-67), amplification of the HER-2/neu oncogene, and accumulation of the p53 protein. After a median duration follow-up period of 74 months, the parameters of tumor diameter > or =20 mm, HER-2/neu oncogene amplification, and p53 mutation were found to be associated with a statistically significant shortened duration of disease-free and overall survival, but not the parameters of tumor grade, hormone receptor levels, or p53 expression. The poor prognosis associated with p53 mutation was observed primarily in patients with a tumor diameter of > or =20 mm. In multivariate analysis, p53 mutation was a risk factor for increased risk of recurrence and death from breast cancer independent of tumor size, hormone receptor levels, HER-2/neu amplification, and MIB-1 expression. We conclude that a relatively simple and rapid single strand conformation polymorphism method of determining p53 mutation status in node-negative breast cancers can provide independent prognostic information.     

Adenosine 3'':5''-cyclic monophosphate induces regulated secretion of tissue-type plasminogen activator and von Willebrand factor from cultured human endothelial cells

The effects of the short-term pre-operative administration of tamoxifen (TAM, 20 mg once daily) on the tumor levels of steroid receptors and the nuclear proliferation Ki-67 antigen, were investigated in 32 elderly patients with hormone-sensitive, operable primary breast cancer by means of fine-needle aspiration biopsy (FNAB). The FNAB smears before (pre-TAM) and after six weeks of treatment (post-TAM) were stained immunocytochemically in order to obtain an H-score for steroid receptors, and the percentage of cellular nuclei containing Ki-67. The mean oestrogen receptor (ER) score between the pre- and post-TAM specimens fell from 181.2 9.7 ( SEM) to 148.1 7.9 (Wilcoxon's matched-pairs signed-rank test, p=0. 01) and there was also a significant decrease in both the mean progesterone receptor (PgR) score (178.4 10.6 vs 148.5 10.6; p=0.01) and mean Ki-67 index (8.2% 1.2 vs 4.9% 0.9; p=0.0002). The reliability of FNAB as a sampling method was checked by comparing the results of the immunocytochemical assay (ICA) of the post-TAM biopsies with those of the immunohistochemical assay (IHA) of the corresponding excised tumors. There was a positive correlation between the ICA and IHA scores: ER (Spearman's correlation coefficient, rho=0.66, p<0.001), PgR (rho=0.84, p<0.001) and Ki-67 (rho=0.96, p<0.001). We conclude that the sequential use of FNAB is a reliable means of assessing the behaviour of within-tumor biomarkers during endocrine therapy.     

Randomized, double-blind, placebo-controlled study of ascorbate on the preventive effect of nitrate tolerance in patients with congestive heart failure

Mitogen-activated protein kinase (MAPK) is a serine-threonine kinase that is activated by various extracellular stimuli. Extracellular signal-regulated kinases (ERK1 and ERK2), an MAPK subfamily, are activated by many oncogenes, such as ras and raf, and they induce cell proliferation. myc is also an oncogene and one of the targets of ERKs. Mutations of ras and overexpression of myc were found in various human cancers, and ERKs were also reported to play a role in carcinogenesis. In this study, we examined 39 biopsy specimens of oral squamous cell carcinoma (OSCC) and 5 of normal gingival mucosa for the expression of ERK protein and the proliferation marker, MIB-1 (Ki-67 antibody). Thirteen OSCC specimens and five normal gingival biopsies were also examined for the expression of ERKs mRNA by in situ hybridization. Double staining for ERKs and MIB-1 was also performed. Histologically, 18 patients (46%) were diagnosed with well-differentiated SCC, 17 (44%) with moderately differentiated SCC, and 4 (10%) with poorly differentiated SCC. The histologic grade correlated with the MIB-1 index. The localization of ERK1 was similar to that of ERK2. Positive signals for ERK proteins were localized in superficial keratinocytes in normal gingival mucosa, whereas these mRNAs were weakly positive in the basal and spinous layer. Basal and suprabasal cells were positive for MIB-1. In well-differentiated and moderately differentiated OSCC, positive signals for ERK mRNA and proteins were found at higher levels than in normal gingival mucosa in keratotic cells around cancer pearls. Some cells showed positive signals for ERKs and MIB-1. Furthermore, most cancer cells in poorly differentiated SCC were positive for both ERK and MIB-1. The histologic grade was statistically related to the percentage of cells positive for both ERK and MIB-1. This suggested that ERKs might be related to proliferation in OSCC.     

Postpartum onset of panic disorder

Cyclin D1 (cycD1) expression was defined immunohistochemically using monoclonal antibody DCS-6 and polyclonal antiserum H-295 in 50 glioma biopsies. The number of positive nuclei was higher for H-295 than for DCS-6, with a ratio of 3:1. The labelling index (LI) was compared to the grade of histological malignancy and to Ki-67 MIB-1 LI. The LI for cycD1 increased with histological malignancy, in parallel with the increase in MIB-1 LI. In most tumours, the maximum LI for cycD1 and MIB-1 were found in the same areas. The mean MIB-1 LI: mean cycD1 LI ratio does not vary in the three grades of astrocytic tumours. However, in this study the correlation between the two LIs was not statistically significant. Staining for cycD1 antigen does not necessarily imply that the gene is overexpressed since other molecular mechanisms can also be responsible for cell cycle deregulation. In invasive areas, the cycD1 LI is frequently higher than in solid tumour, either because more tumour cells are positive or because reactive astrocytes and activated microglia express cycD1. The relative contribution of neoplastic and reactive cells remains to be defined.