Immunohistochemical evaluation of bcl-2, ER-alpha,caspase -3, -8, -9, PCNA and Ki-67 expressions in canine mammary carcinomas

We investigated the expression of bcl-2, estrogen receptor alpha (ER-alpha), caspase-3, ?8, ?9, proliferating cell nuclear antigen (PCNA) and Ki-67 in canine mammary carcinomas. We used 65 paraffin embedded and re-diagnosed archival canine mammary tumor samples to which we applied the routine streptavidin-biotin-peroxidase technique. Seventeen cases were re-diagnosed as tubulopapillary carcinoma, 31 were re-diagnosed as complex carcinoma and 17 were re-diagnosed as carcinosarcoma. Differences of expression of bcl-2 and PCNA were statistically significant according to tumor type. Differences in expression of ER-alpha, caspase-3, ?8, ?9 and Ki-67 were not statistically significant. Differences of expression of bcl-2 and PCNA were statistically significant compared to ER-alpha, caspase-3, ?8, ?9 and Ki-67 in carcinosarcomas. We report the prognostic significance of bcl-2 and PCNA expression in canine mammary carcinosarcomas.     

Immunohistochemical analysis for cell proliferation-related protein expression in small cell carcinoma of the esophagus; a comparative study with small cell carcinoma of the lung and squamous cell carcinoma of the esophagus

Small cell carcinoma is a rare neoplasm in the esophagus. To evaluate cell proliferation activity and its underlying mechanisms in this tumor, we examined immunohistochemically 5 cases of small cell carcinoma of the esophagus (SCCE) for expressions of tumor suppressor proteins, oncoproteins and cell proliferation markers including p53, p21WAF1/CIP1, retinoblastoma (Rb) protein, bcl-2, Ki-67 and PCNA, and compared the results with those of 5 cases of small cell carcinoma of the lung (SCCL) and 10 cases of squamous cell carcinoma of the esophagus (SQCE). The prevalence and labeling index of p53-immunoreactivity tended to be higher in SCCE (4/5; 56.6%) and SCCL (4/5; 79.9%) than in SQCE (6/10; 48.8%). Expression of p21WAF1/CIP1 was observed in 2 of 10 cases of SQCE. In contrast, its expression could not be detected in any cases of SCCE and SCCL examined. Expression of Rb protein was observed in 9 out of 10 cases of SQCE, but not in any cases of SCCE and SCCL. SCCE and SCCL showed more frequent and intense immunoreactivity for bcl-2 than SQCE. In expression of cell proliferation markers (Ki-67 and PCNA), no remarkable difference was observed among SCCE, SCCL and SQCE. These results suggest that SCCE and SCCL could share some genetic alternations including mutation of p53, loss of Rb gene and overexpression of bcl-2, and these may be related to the similar biological potentials between the two. Furthermore, SCCE was different from SQCE in expression of Rb protein and bcl-2, and these two types of esophageal carcinoma could arise through different molecular mechanisms.     

Prediction of aggressiveness of gastrointestinal stromal tumours based on immunostaining with bcl-2, Ki-67 and p53

OBJECTIVE: While the use of fine needle aspiration (FNA) in the diagnosis of gastrointestinal stromal tumours (GISTs) is well-established, it can be difficult to predict the prognosis of GIST based on morphology alone. The objective of the current study was to determine if expression of bcl-2, Ki-67 and p53 correlated with the outcome of GISTs based on cytological material. METHODS: Cell-blocks from 14 GISTs diagnosed by FNA were retrieved. Immunostaining was performed with antibodies against bcl-2, Ki-67 and p53. All cytological diagnoses were confirmed by positive immunostaining with c-kit and/or subsequent histological evaluation. Positivity for bcl-2, Ki-67 and p53 was defined as the presence of > or =10% cytoplasmic staining, > or =5% nuclear staining and > or =5% nuclear staining respectively. RESULTS: The 14 patients consisted of seven males and seven females with a mean age of 58 years. The average follow-up interval was 46 months. Six had a benign course and eight developed recurrences/metastases. Thirteen (93%) cases showed positive staining for bcl-2. Positive Ki-67 and p53 staining was noted in one (7%) and seven (50%) cases respectively. The difference in staining for p53 between aggressive and non-aggressive GISTs was statistically significant. No statistically significant difference was noted for bcl-2 staining or Ki-67 labelling index between the two groups. CONCLUSIONS: According to our observations, p53 immunostaining may be useful in predicting the outcome of GIST diagnosed by FNA; Ki-67 and bcl-2 are not useful as prognostic markers for GIST in FNA specimens.     

Expression of p120, Ki-67 and PCNA as proliferation biomarkers in imprint smears of prostate carcinoma and their prognostic value

The cell proliferation markers p120, Ki-67 and proliferating cell nuclear antigen (PCNA) recognize nuclear antigens. The expression of these proteins by immunostaining methods was reported to be of value in determining the prognosis of patients with malignant diseases. In this study, we evaluated the prognostic significance of the expression of nuclear antigens p120, PCNA and Ki-67 in prostate cancer and compared the results with other prognostic factors. Imprint smear samples obtained from 70 patients immediately after radical prostatectomy for prostatic carcinoma were immunostained with monoclonal antibodies against p120, Ki-67 and PCNA. The immunostaining results were correlated with Gleason score, tumour differentiation, stage and prostatic specific antigen (PSA) levels. Our findings demonstrate that p120, Ki-67 and PCNA expression in prostatic carcinoma smears, correlated significantly with the degree of Gleason score (P < 0.001). When combining p120, Ki-67 and PCNA positivity with tumour differentiation there was a significant association among these parameters (P < 0.001). Overexpression of p120, Ki-67 and PCNA, was also associated with increased PSA serum levels (>4 ng/ml) (P < 0.001). The distribution of p120, Ki-67 and PCNA expression in prostate carcinomas was not statistically significant for Ki-67 (P = 0.69) and p120 (P = 0.22) but was significant for PCNA (P < 0.001) as far as the histological stage (T2a, T2b, T2c, T3a). P120, Ki-67 and PCNA expression had significant prognostic value for disease-free survival. Our results conclude that nuclear antigens p120, Ki-67 and PCNA appear to be additional markers in the field of prognosis of prostatic carcinoma.     

Ultrasound-guided fine needle aspiration cytology of parathyroid lesions. A review of 72 cases

OBJECTIVE: To elucidate the specific cytomorphologic patterns and diagnostic pitfalls in fine needle aspiration cytology (FNAC) of parathyroid lesions. STUDY DESIGN: Seventy-two cases of surgically excised and pathologically verified hyperparathyroidism (20 cases of parathyroid hyperplasia, 51 of parathyroid adenoma and one of parathyroid carcinoma) received preoperative, ultrasound-guided FNAC examination for enlarged parathyroid glands. The smears were reviewed and analyzed. RESULTS: Parathyroid lesions were diagnosed cytologically in 60 cases (83.3%). The presence of colloidlike substance, macrophages or follicular structures in smears led to six cases (8.3%) being misinterpreted as thyroid lesions. The cellularity of the smears was insufficient for interpretation in six cases (8.3%); however, two of these cases were diagnosed by determination of parathyroid hormone (PTH) levels in the fluid. Parathyroid hyperplasia had more tightly cohesive cell clusters with monomorphism, while parathyroid adenoma had more dispersed or loosely cohesive cells with pleomorphism and anisokaryosis. High PTH concentration in an aspirate was noted in all four cases of cystic lesions.     

Changes in gene expression in the course of proliferative processes in the parathyroid gland

The aim of this study was to investigate the changes in expression pattern of the most important genes connected with apoptosis in proliferative apoptotic lesions (hyperplasia, adenoma), applying cDNA microarray technique, in order to promote the possible diagnostic or therapeutic utilisation of any difference in gene expression compared to the healthy (normal) parathyroid gland. Samples were taken from surgically removed 2 hyperplasias, 2 adenomas and 2 normal parathyroid glands. The Apoptosis Gene Array (Superarray) was used. This contains 112 genes, in tetraspot arrangement. The probes measured 250-600 base pairs. Streptavidin was bound to the array. CDP Star TM chemiluminescent substrate was used for detection. The samples deriving from hyperplasia or adenoma were compared to samples from normal parathyroid glands. The following genes were overexpressed in both hyperplasia and adenoma: CHEK1, ATM, BCL-XL, FAS, TNF, cIAP1, TRAIL, FADD, CASP 4,5,6,8, CD120b, CD137, LTA, TANK, TARF2, CAD, LIGHTR, DR3LG. CASP1,10, BFAR, BOD, BCL2L2, TRANCE were underexpressed in both hyperplasia and adenoma. Genes overexpressed only in hyperplasia were: MDM2, MCL1, BCL2A1, BLK, RIPK2, CD40LG, TRAF5, HUS1, BNIP3. Underexpressed only in hyperplasia: BOK, CIDEA, TRAF1, TRIP. Overexpressed only in adenoma: APOLLON, RIPK1, LTB, LTBR, CASP2,13, cIAP2, CIDEB. Underexpressed only in adenoma: TRAF4 and FASLG. Overexpresion or underexpression meant 1.5-fold difference from normal average values. As a result of this study, both pro-apoptotic and antiapoptotic genes were identified in hyperplasia and adenoma of the parathyroid gland. It seems that increased proliferation is connected also with increased apoptotic activity, but tumor cell candidates are able to survive, by activation of signal pathways resulting in overexpresion of anti-apoptotic genes.     

Cell proliferation in prostatic carcinoma: comparative analysis of Ki-67, MIB-1 and PCNA

Summary Antibodies to assess the proliferative index of tumours are being increasingly employed together with established markers for prognostic evaluation. This study set out to compare three cell proliferation markers, Ki-67, MIB-1 and PCNA, utilizing a semiquantitative method of assessment, in 20 human prostatic carcinomas. The streptavidin-biotin immunostaining system was used for the monoclonal antibodies MIB-1 and PCNA and an indirect immunoperoxidase assay for the monoclonal antibody Ki-67. Significant correlations were found between the expression of Ki-67 in frozen tissues and MIB-1 in formal saline-fixed wax-embedded tissues (p = 0.0003); between Ki-67 and PCNA expression in Bouin's-fixed tissues (p </ 0.0001); and MIB-1 (formalin-saline-fixed tissues) and PCNA (Bouin's-fixed tissues) (p </ 0.0001). A more intense nuclear staining pattern with less heterogeneity was observed for MIB-1 compared with PCNA, suggesting the antibody of choice, on formal saline-fixed tissues, is MIB-1, which closely correlated with Ki-67, a marker we have previously shown to be of prognostic value in prostatic carcinoma.     

Role of apoptosis and proliferation in differentiation of human retina,regio olfactoria and regio respiratoria

Apoptosis and proliferation are very important processes during central nervous system development. In our study we concentrated on human fetus retina, regio respiratoria and olfactoria. The aims of our work were to evaluate a proliferative activity proved by PCNA and KI-67 gene expressions, to measure an expression of anti-apoptotic gene bcl-2, and finally to compare levels of apoptosis and proliferation in the areas investigated. Altogether we studied 29 fetuses between the 5th to 13th week of gestation (WG). Parallel sections were elaborated by using standard methods of immunohistochemistry for the detection of proteins PCNA, Bcl-2 and Ki-67. Apoptotic cells were detected by two methods: TUNEL (for the detection of DNA fragmentation) and Apostain (for evidence of ssDNA). The results of semiquantitative evaluation show that expressions of proteins PCNA and KI-67 begins after the 6th WG, from the 8th to 13th WG they are very high in all investigated areas. Occurrence of apoptotic cells in regio respiratoria is sporadic between the 5th to 9th WG, while later it is widely spread. Apoptosis in regio olfactoria appears after the 9th WG and it is also very extensive. In the retina we found a higher frequency of apoptotic cells only in the 11th to 13th WG. The results of both methods for detection of apoptosis were concordant. Anti-apoptotic gene bcl-2 was detectable in all examined areas, but it followed no orderly pattern. We conclude, that the expression of apoptosis increases with the age from the 7th to 13th WG, and that both apoptosis and proliferation participate in the morphogenesis of fetuses between the 5th to 13th WG.     

Proliferative activity as detected by immunostaining with Ki-67 and proliferating cell nuclear antigen in benign and malignant epithelial lesions of the human parotid gland

OBJECTIVE: A retrospective immunohistochemical study of parotid gland lesions was designed to evaluate the diagnostic and prognostic value of the proliferating cell nuclear antigen (PCNA) and Ki-67 with monoclonal antibodies PC 10 and MIB-1, respectively. STUDY DESIGN: Tissue samples comprised normal parotid gland (N, n = 10), chronic sialadenitis (CS, n = 8), Warthin's tumor (W, n = 10), benign pleomorphic adenoma (BPA, n = 8), mucoepidermoid carcinoma (MEC, n = 13), carcinoma in pleomorphic adenoma (CPA, n = 8) and adenoid cystic carcinoma (ACC, n = 12). The morphometric parameters for PCNA and MIB-1 comprised the PI and MI labelling indices (the numerical percentage of positive nuclei), NAP and NAM (the numerical density of positive nuclei), and NPI and NMI (volume corrected index). RESULTS: The values of MIB-1 parameters increased progressively in benign lesions in comparison with the N group and in malignant neoplasms in comparison with nonneoplastic groups and benign lesions. Values for all parameters in BPA were significantly lower than those in malignant groups. Spearman rank correlation analysis showed a highly positive correlation between the morphometric parameters and severity of the lesions. The mean values of MI and NMI were significantly higher in patients who died of the malignant tumors than in those who survived. The same quantitative parameters for PCNA did not differ significantly from those obtained for MIB-1 and showed similar trends. CONCLUSION: PCNA and MIB-1 indices are reliable markers for discriminating between benign and malignant tumors of the parotid gland, and the parameters PI, MI, NPI and NMI may have prognostic applications.     

Detection of cancer clones in human colorectal adenoma as revealed by increased DNA instability and other bio-markers

An immunohistochemical differential staining of cancerous cells with anti-cytidine antibody after denaturation of nuclear DNA by acid hydrolysis with 2N HCl at 30 degrees C for 20 min (DNA-instability test) has been used as a marker for malignancy. The test was applied to bioptic tissues of human colorectal polyps assessed histopathologically as hyperplastic polyp (11 cases), tubular adenoma of mild (68 cases), moderate (102 cases), and severe (46 cases) dysplasia, and adenocarcinoma (30 cases). The serial sections of the same tissues were also subjected to immunohistochemical staining for Ki67, p53, DNA-fragmentation factor 45 (DFF45) and vascular endothelial growth factor (VEGF). The DNA-instability test was positive in 30 (100%) adenocarcinoma cases, 46 (100%) severe dysplasia adenoma cases, 36 (35.29%) moderate dysplasia adenoma cases, and 8 (11.76%) mild dysplasia adenoma cases, indicating malignancy. All hyperplastic polyps were negative to the DNA-instability test. Furthermore, the percentage of glands positive in the DNA-instability test steadily increased in going from mild (10%), to moderate (35%), to severe (100%) dysplasia, and adenocarcinoma (100%). All other biological markers tested in the present study showed significantly higher values in those adenoma glands that were positive to the DNA-instability test, irrespective of the dysplasia grade, as compared to the markers in the adenoma glands that were negative to DNA instability testing. Furthermore, the former values were comparable to those in adenocarcinoma. The results indicate that cancer cell clones are already present at the adenoma stages showing positivity to DNA instability testing, enhanced proliferative activity, p53 mutation and induction of DFF45 and VEGF, at a time when the degree of morphological atypia are not yet large enough for them to be identified as cancer. These factors promote cancer cell proliferation, produce heterogeneous subclones due to DNA instability, enhance their survival by escaping apoptosis, and provide abundant nutrients by neovascularization during the early-stage progression of colorectal cancer.     

Silencing of RHEB inhibits cell proliferation and promotes apoptosis in colorectal cancer cells via inhibition of the mTOR signaling pathway

Colorectal cancer (CRC) is commonly known as one of the most prominent reasons for cancer-related death in China. Ras homolog enriched in brain (RHEB) and the mammalian target activity of rapamycin (mTOR) signaling pathway were found correlated with CRC, but their specific interaction in CRC was still to be investigated. Therefore, we explored whether RHEB gene silencing affected the cell proliferation, differentiation, and apoptosis by directly targeting the mTOR signaling pathway in cells previously harvested from CRC patients. A microarray analysis was subsequently conducted to investigate the relationship between RHEB and mTOR. Eighty-three adjacent normal tissues and CRC tissues were selected. Immunohistochemistry was carried out to detect the positive expression rates of RHEB and Ki-67 in the CRC tissues. Cells were then transfected with different siRNAs to investigate the potential effects RHEB would have on CRC progression. The expressions of RHEB, 4EBP1, ribosomal protein S6 kinase (p70S6K), proliferating cell nuclear antigen (PCNA), B cell lymphoma 2 (bcl-2), and bcl-2-associated X protein (bax) were determined and then the cell cycle, cell proliferation, and apoptotic rate were also measured. We identified RHEB and mTOR as upregulated genes in CRC. Cells treated with RHEB silencing showed a decreased extent of mTOR, p70S6K, 4EBP1 phosphorylation and expression of RHEB, Ki-67, mTOR, p70S6K, 4EBP1, bcl-2, and PCNA as well as decreased activity of cell proliferation and differentiation; although, the expression of bax was evidently higher. Collectively, our data propose the idea that RHEB gene silencing might repress cell proliferation and differentiation while accelerating apoptosis via inactivating the mTOR signaling pathway.     

Some oncogene and tumour suppressor gene protein products expression in B-cell chronic lymphocytic leukaemia

The expression of Bcl-2, P53 proteins and known markers of proliferation, namely proliferating cell nuclear antigen (PCNA) and Ki67, in 29 patients with B-cell chronic lymphocytic leukaemia (B-CLL) was investigated. All leukaemic patients were classified, and immunophenotyped by the two-colour immunofluorescence method with the use of fluorocytometry. B-CLL was heterogeneous in the range of biological parameters of tumour cells. B-CLL patients manifested 34% positive Ki67 and 61% PCNA expression, whereas Bcl-2 and P53 positivity was 81% and 42%, respectively. The level of intracellular expression of Bcl-2 and P53 proteins did not depend on the stage of disease estimated by routine methods. Ki67 and PCNA expression was significantly higher in B-CLL patients with more advanced stages of the disease. A statistically significant correlation was established between their mutual expression.     

Expression of parathyroid hormone-related protein (PTHrP) in parathyroid tissue under normal and pathological conditions

Parathyroid hormone-related protein (PTHrP), a factor responsible for malignancy associated hypercalcemia, plays a physiological roles such as bone development and placental calcium transport. The expression of PTHrP in adult human parathyroid tissues under normal and pathological conditions was analyzed. By immunohistochemistry, PTHrP expression was detected in 86% of normal parathyroid (12/14 cases), 74% of adenomas (14/19) and 89% of hyperplasia secondary to chronic renal failure (16/18). PTHrP protein was observed mainly in the cytoplasm of oxyphil cells, consistent with the localization of its mRNA demonstrated by in situ hybridization. The rate of PTHrP-positive cells was higher in areas consisting of oxyphil cells than in those of non-oxyphil cells, regardless of whether the parathyroid was normal or pathological. In the normal parathyroid, an age-related increase in PTHrP expression was observed with a relative increase in oxyphil cells, reflecting aging and deterioration of parathyroid tissue. In adenoma, cases with a predominance of oxyphil cells expressed PTHrP, whereas clear cell adenoma did not. In secondary hyperplasia, the rate of PTHrP-expressing cells was higher than in normal parathyroid or adenoma, with varying levels of expression among nodules. We speculate that PTHrP could act through the paracrine/autocrine mechanism to regulate proliferation and differentiation of normal and neoplastic parathyroid cells.     

Are biomarkers correlated with recurrence patterns in patients with resectable gastric adenocarcinoma

The aims of this study are to analyze the failure patterns in radical resected gastric adenocarcinoma, and to evaluate the correlation between recurrence patterns and potentially prognostic factors, including clinical pathological characteristic and biomarkers. Between Jan 2004 and Jun 2006, 84 patients were enrolled into the database analysis, including 8 with clinical stage I, 20 with clinical stage II, 21 with clinical stage IIIA, 22 with clinical stage IIIB and 13 with clinical stage IV, male 61 and female 23. The collected biomarkers including: preoperative tumor markers: CEA, AFP, CA199, CA50, CA72-4 and CA24-2; postoperative immunohistochemical (IHC) markers: Bax, Bcl-2, P27, CyclinD1, TOPO2, MDR, GST-π, Ki67, epidermal growth factor receptor (EGFR), P21, P53, proliferating cell nuclear antigen (PCNA), C-myc and Neu. Three-year local control rate (LCR), disease-free survival (DFS) and over-all survival (OS) were 66, 61 and 64% respectively. Logistic regression analysis showed cyclinD1 and CEA were correlated with prognosis; cyclinD1, CEA were correlated with loco-regional recurrence; PCNA was correlated with remote metastasis; bcl-2, ki67, c-myc2 and Neu were correlated with lymph node metastasis. The present study indicate that patterns of recurrence are variable and may be associated with specific biomarkers, in addition, high level of CEA and low-expressed of cyclinD1 resulted in poor prognosis.     

Cyclin D1 Immunoreactivity in Meningiomas

Objective Cyclin D1 is an important nuclear protein required for progression of cells through the G1 phase of the cell cycle. The proliferative potential of meningiomas has been studied using various proliferative markers. However, there have been only few published studies evaluating Cyclin D1 immunoreactivity in meningiomas. Purpose of the study The aim of our study was to analyze the Cyclin D1 expression in meningiomas and correlate it both with proliferation markers Ki67 and PCNA, and with meningiomas of WHO grade. Material and methods We evaluated immunoreactivity for proliferative markers (Cyclin D1, Ki-67, and PCNA) in a consecutive series of 64 meningioma samples obtained from patients who underwent surgical resection because of cerebral or spinal meningiomas. Immunohistochemical staining with Ki-67, PCNA, and Cyclin D1 was performed using the microwave processing procedure and LSAB+ methodology. The number of positive cells for each antibody has been determined and shown in percentage in relation to 1000 counted cells. Results All meningioma samples showed immunostaining for Ki-67, PCNA, and Cyclin D1 antibodies. The Cyclin D1 scores exhibited a close correlation with Ki-67 and PCNA immunostaining (P < 0.01). Some meningiomas (15 cases) showed a combination of nuclear and cytoplasmatic (fine granular) Cyclin D1 immunoreactivity. All proliferative indexes have been in positive correlation with meningioma grade. Conclusion Our comparative study of proliferative markers in meningiomas demonstrated Cyclin D1 as a very useful proliferative marker in meningiomas.     

肺癌中增殖细胞核抗原表达、DNA含量测定及其与预后的关系

本文采用ＳＰ免疫组织化学方法对６７例肺癌进行增殖细胞核抗原染色、与图像分析技术测定ＤＮＡ含量作相关性分析,探讨其在判断肺癌预后方面的意义。结果：（１）ＰＣＮＡ阳性表达强度与预后明显相关。（２）肺癌组织中ＰＣＮＡ表达强度与ＤＮＡ含量之间存在较好的相关性。（３）肺癌组织中多倍体随ＰＣＮＡ表达增强而增多,二倍体则随ＰＣＮＡ表达增强而减少。说明用简单的免疫组织化学方法检测肺癌组织中ＰＣＮＡ,以此来观察肿瘤细胞增殖活性,可作为判断肺癌患者预后的指标之一,易被广大基层单位推广应用     

Emerging role of p53, bcl-2 and telomerase activity in Egyptian breast cancer patients

Apoptotic cell death represents an important mechanism for the precise regulation of cell numbers, and a defense mechanism against tumor cells. Both bcl-2 and mutant p53 gene products have been involved in apoptotic pathways. On the other hand, cell proliferation capacity and tumorgenesis have been controlled by telomerase. The purpose of our study is to assess the prognostic significance of additional markers implicated in apoptosis and tumorgenesis. Fifty-one fresh tissue samples of primary breast carcinoma and 26 tissue samples of benign breast lesions were included in this study. Expression of bcl-2 in cell lysates and mutant p53 protein in nuclear fraction were measured by Oncogene Science EIA procedures. Telomerase activity was analyzed using the Telomerase-PCR-ELISA based on the TRAP (telomerase repeat amplification protocol) method. On the same specimens, steroid hormone receptors (ER and PgR) were measured in cytosol fraction using Abbott EIA assays. In addition, information regarding surgical-pathological features of the tumor was obtained. Univariate and Multivariate analysis was done to identify variables predictive of poor prognosis. Significant expression of bcl-2, mutant p53 proteins and relative telomerase activity were observed in malignant cases when compared to benign ones. Univariate analysis revealed significant association in the level of both mutant p53 and relative telomerase activity with tumor size and disease recurrence. Moreover, telomerase activity was significantly expressed in late stages than early ones. Multivariate analysis revealed that bcl-2, mutant p53, telomerase activity, PgR and age were independent prognostic factors. Among a panel of molecular genetic factors investigated, mutant p53 and relative telomerase activity were strongly associated with disease recurrence; hence they exert a significant prognostic role in breast cancer.     

PCNA/cyclin expression and BrdU uptake define different subpopulations in different cell lines.

Monoclonal antibodies (MAb) to a 36 KD protein, proliferating cell nuclear antigen (PCNA/cyclin), have been previously shown to be capable of identifying proliferating cells in vitro as well as in alcohol-fixed, paraffin-embedded tissue specimens. The routine use of these anti-PCNA/cyclin MAb in investigative studies and in diagnostic pathology requires a clearer understanding of the distribution of PCNA/cyclin in the different cell populations found in tissue specimens. We therefore compared the ability of MAb to three nucleus-associated proliferation markers (MAb 19A2 to PCNA/cyclin; Ki-67 to an undefined proliferation-related marker; BU-1 to 5'-bromodeoxyuridine (BrdU) incorporated into DNA) to identify the proliferating cell fraction of various cells in vitro. The cell lines were chosen to represent a spectrum of proliferation rates (high to low) and cell lineage (mesenchymal vs epithelial, non-transformed vs malignant): (a) HeLa and A-431 (two malignant carcinoma cell lines with high proliferation rates); (b) SK-5 (a non-transformed fibroblast cell line with a low proliferation rate); (c) HUVE (a non-transformed human umbilical vein endothelial cell line with a low proliferation rate). Single and double labeling immunofluorescence studies were performed after uniform 1-hr incubations with BrdU. Comparison of the overlapping distributions of detectable PCNA/cyclin expression and BrdU incorporation demonstrated substantial qualitative and quantitative differences between the different cell lines. In two of the four cell lines (HeLa, A-431) the BrdU staining distributions formed inclusive subsets of the PCNA-positive cell populations. In the HUVE cell line the two populations overlapped incompletely. In one cell line, SK-5, the two populations were mutually exclusive. MAb Ki-67 demonstrated a pattern in the SK-5 cell line that was strongly predictive of PCNA positivity, while showing no associated patterns in the other three cell lines. We conclude that PCNA/cyclin expression detected by MAb may define different cell subpopulations in different cell types relative to those incorporating BrdU or expressing the target antigen for Ki-67. This has implications for the clinical study of mixed cell populations using these antibodies.     

Expression and significance of cell immunohistochemical markers (HHF-35, CD-31, Bcl-2, P-53 and apopDETEC) in hypertrophic cardiomyopathy

There are several hypotheses concerning the pathogenesis of hypertrophic cardiomyopathy (genetic, ischaemic, immune, inflammatory and apoptosis induction). We have studied three types of cardiomyopathy in order to observe the expression and assess the significance of different immunohistochemical markers (muscular actin, CD-31, proliferation cell nuclear antigen -PCNA-, Ki-67, and markers related with programmed cell death, bcl-2, p-53 and apopDETEC). We studied different microscopic (haematoxylin-eosin and Masson's thrichrome) and immunohistochemical parameters (streptavidin-biotin-peroxidase and "in situ" hybridisation) of forty cases: ten each of hypertensive hypertrophic cardiomyopathy, essential hypertrophic cardiomyopathy, hypertrophic cardiomyopathy in patients treated with chemotherapy and morphologically "normal" hearts. Our findings point to an absence of structural marker expression (actin and CD-31) in cases of hypoxic damage. The distribution and intensity of apoptosis markers, a seen by "in situ" hybridisation were irregular, and the rest of the markers studied showed negative results, with the exception of acridin orange (a marker of hypoxic damage). In our opinion, the above immunohistochemical markers, especially actin and CD-31, could be used for differentiating hypoxic lesions in these three types of cardiomyopathy. Moreover, it is difficult to know the significance of the apoptosis markers, because the autolysis process produces cross reactions with false positive results. We think that there is a need for new studies on DNA breakdown processes during the post-mortem interval. To avoid autolysis problems the post-mortem material needs to be as fresh as possible.     

bel-2 protein in breast cancer cells obtained by fine needle aspiration (FNA): a preliminary report

The bcl-2 protein plays a role in the regulation of programmed cell death (PCD), overriding apoptosis. Its expression has been reported in breast ductal cells, where it is believed to be involved in the hormonal regulation of hyperplasia and involution. to date, bcl-2 gene product has not been investigated on breast cancer FNA. the expression of bcl-2 protein was evaluated using an immunoalkaline phosphatase technique in 54 pre-operative breast cancer aspirates and in paraffin-embedded sections from 20 matched surgical specimens. A high rate of bcl-2 protein expression was found on FNA samples (65%) and on the corresponding tissue sections (60%); there was a nearly absolute concordance in the two specimens, with 19/20 (95%) cases showing a concordant staining. These findings concur with the view that bcl-2 gene is frequently expressed in breast cancer, possibly through a hormonal-dependent pathway.