Bmi-1 Immunohistochemical Expression in Endometrial Carcinoma is Correlated with Prognostic Activity.

Background and objectives: B-lymphoma Mo-MLV insertion region 1 (Bmi-1) is a stem cell factor that is overexpressed in various human cancer tissues. It has been implicated in cancer cell proliferation, cell invasion, distant metastasis, and chemosensitivity, and is associated with patient survival. Several reports have also identified Bmi-1 protein overexpression in endometrial carcinoma; however, the relationship between Bmi-1 expression and its significance as a clinicopathological parameter is still insufficiently understood. Accordingly, the present study aimed to clarify whether immunohistochemical staining for Bmi-1 in human endometrial carcinoma and normal endometrial tissues can be used as a prognostic and cell proliferation marker. Materials and Methods: Bmi-1 expression was assessed in endometrioid carcinoma (grade 1-3) and normal endometrial tissues (in the proliferative and secretory phases) by immunohistochemistry; protein expression was evaluated using the nuclear labeling index (%) in the hot spot. Furthermore, we examined other independent prognostic and proliferation markers, including the protein levels of Ki-67, p53, and cyclin A utilizing semi-serial sections of endometrial carcinoma tissues. Results: The expression of the Bmi-1 protein was significantly higher in all grades of endometrial carcinoma than in the secretory phase of normal tissues. Moreover, Bmi-1 levels tended to be higher in G2 and G3 tissues than in G1 tissue, without reaching significance. Bmi-1 expression showed no notable differences among International Federation of Gynecology and Obstetrics (FIGO) stages in endometrial carcinoma. Furthermore, we observed a significant positive relationship between Bmi-1 and Ki-67, cyclin A, or p53 by Spearman's rank correlation test, implying that high Bmi-1 expression can be an independent prognostic marker in endometrial carcinoma. Conclusions: Our study suggests that Bmi-1 levels in endometrial carcinoma tissues may be useful as a reliable proliferation and prognostic biomarker. Recently, the promise of anti-Bmi-1 strategies for the treatment of endometrial carcinoma has been detected. Our results provide fundamental data regarding this anti-Bmi-1 strategy.

Divergent immunohistochemical expression of CD21 and CD23 by follicular dendritic cells with increasing grade of follicular lymphoma.

BACKGROUND: Ultrastructural and immunohistochemical differences have been described in FDCs of primary and secondary follicles, illustrating the highly compartmentalized structure of lymph follicles. Differences in FDC immunophenotype in different grades of FL may reflect some parallelism between reactive and neoplastic conditions in terms of FDC-B cell interaction and may be used as a valuable additional tool for grading FL. METHODS: A total of 60 paraffin blocks from patients with follicular lymphoma, 30 cases each of grade 1 and 3, were retrieved from our archive. Immunohistochemical analysis was carried out for CD21, CD23, cyclin A, and Ki-67. RESULTS: Our study demonstrates that during evaluation, six patterns of FDC distribution were distinguished. The intensity of stain for CD21 was not statistically significant in grade 1 and grade 3 FL (p = 0.340). In contrast, grade 3 FLs exhibited a significant decrease of CD23 expression by the FDCs (p < 0.001). By CD21 stain, there was no significant difference in the distribution of pattern 1 in grades 1 and 3 (p = 0.098). In contrast, in grade 3, this pattern was significantly less observed by CD23 stain (p = 0.016). The same was observed for pattern 2 for CD21 (p = 0.940) and CD23 (p = 0.010) and pattern 4 for CD21 (p = 0.305) and CD23 (p = 0.005), respectively. Distribution of pattern 5 was significantly different between grades 1 and 3 both for CD21 (p = 0.005) and CD23 (p < 0.001). Distribution of patterns 2 and 6 was not significantly different between grades 1 and 3 for CD21 and CD23. The values of cyclin A and Mib-1 were also significantly different between grades 1 and 3 (p < 0.001). CONCLUSIONS: The observed patterns of FDCs lead us to believe that similar to reactive lymph node follicles, neoplastic follicles in FL, at least in early stages, have an organized structure. Hypothetically, with CD21, CD23, and cyclin A immunohistochemistry, the sequence of events in FL progression may be traced.

Influence of fixation method and duration of archiving on immunohistochemical staining intensity in embryonic and fetal tissues.

INTRODUCTION: In this study we detail the effect of different fixation agents and the duration of storage has on the immunohistochemical staining positivity of samples of archival embryonic and fetal tissues. MATERIALS AND METHODS: The samples were stained by indirect two-step immunohistochemistry (IHC) method for Ki-67, cyclin A and ß-actin. RESULTS: Irrespective of the length of tissue archiving, tissue fixation with 10% neutral buffered formalin had better IHC intensity results in all cases when compared to methacarn-fixed tissues. In the case of ß-actin, this difference was statistically significant, while differences in Ki-67 and cyclin A were not. The second aspect studied was which effect tissue block archiving duration has on the IHC reactivity. We demonstrated a statistically significant decrease in IHC positivity for all studied antigens between the samples that were archived for 10-19 or 20-45 years, regardless the fixative solution. CONCLUSION: To the best of our knowledge, the influence that the duration of tissue block archiving has on IHC positivity in human embryo and fetal tissue material has not yet been studied. Although the causes of the IHC positivity decline in archived tissue blocks are not well understood, a possible decrease in IHC over time should be considered, particularly in retrospective studies.

CD36 Enhances Vascular Smooth Muscle Cell Proliferation and Development of Neointimal Hyperplasia.

Objective- Dysregulated proliferation of vascular smooth muscle cells (VSMC) plays an essential role in neointimal hyperplasia. CD36 functions critically in atherogenesis and thrombosis. We hypothesize that CD36 regulates VSMC proliferation and contributes to the development of obstructive vascular diseases. Approach and Results- We found by immunofluorescent staining that CD36 was highly expressed in human vessels with obstructive diseases. Using guidewire-induced carotid artery injury and shear stress-induced intima thickening models, we compared neointimal hyperplasia in Apoe-/-, Cd36-/- /Apoe-/-, and CD36 specifically deleted in VSMC (VSMC cd36-/-) mice. CD36 deficiency, either global or VSMC-specific, dramatically reduced injury-induced neointimal thickening. Correspondingly, carotid artery blood flow was significantly increased in Cd36-/- /Apoe-/- compared with Apoe-/- mice. In cultured VSMCs from thoracic aorta of wild-type and Cd36-/- mice, we found that loss of CD36 significantly decreased serum-stimulated proliferation and increased cell populations in S phase, suggesting that CD36 is necessary for VSMC S/G2-M-phase transition. Treatment of VSMCs with a TSR (thrombospondin type 1 repeat) peptide significantly increased wild-type, but not Cd36-/- VSMC proliferation. TSR or serum treatment significantly increased cyclin A expression in wild-type, but not in Cd36-/- VSMCs. STAT3 (signal transducer and activator of transcription), which reportedly enhances both VSMC differentiation and maturation, was higher in Cd36-/- VSMCs. CD36 deficiency significantly decreased expression of Col1A1 (type 1 collagen A1 chain) and TGF-ß1 (transforming growth factor beta 1), and increased expression of contractile proteins, including calponin 1 and smooth muscle α actin, and dramatically increased cell contraction. Conclusions- CD36 promotes VSMC proliferation via upregulation of cyclin A expression that contributes to the development of neointimal hyperplasia, collagen deposition, and obstructive vascular diseases.

miRNA-34a promotes proliferation of human pulmonary artery smooth muscle cells by targeting PDGFRA.

OBJECTIVES: Pulmonary arterial hypertension (PAH) is a fast progressing vascular disease characterized by uncontrolled cell proliferation of pulmonary artery smooth muscle cells (PASMCs). Some studies have suggested that PAH and cancers share an apoptosis-resistant state, featuring excessive cell proliferation. The miR-34 family consists of tumour-suppressive miRNAs, and its reduced expression has been reported in numerous cancers; however, its role in hypoxia-induced PAH has not been previously studied. MATERIALS AND METHODS: miR-34 family expression was evaluated in a rat model with hypoxia and in cultured hypoxic PASMCs, using real-time quantitative PCR (RT-qPCR). Function of miR-34 family was assessed by transfecting miR-34 mimics and inhibitors. Dual luciferase reporter gene assays, RT-qPCR and Western blotting were performed to validate target genes of miR-34. RESULTS: Significant down-regulation of miR-34a in hypoxic lung tissue, pulmonary artery and PASMCs was identified and then effects of miR-34a in modulating cell proliferation in human pulmonary artery smooth muscle cells (hPASMCs) was investigated in vitro. Reduction of miR-34a levels in hPASMCs caused increased proliferation and these effects were reversed by overexpression of miR-34a. miR-34a overexpression down-regulated platelet-derived growth factor receptor alpha (PDGFRA) expression, which is a key factor in PAH development. These results suggest that miR-34a is a potential regulator of proliferation in PASMCs, and that it could be used as a novel treatment strategy in PAH.

Autofluorescence-Directed Confocal Endomicroscopy in Combination With a Three-Biomarker Panel Can Inform Management Decisions in Barrett's Esophagus.

OBJECTIVES: Barrett's esophagus (BE) surveillance with white-light endoscopy and quadrantic biopsies (Seattle protocol) is resource intensive and limited by sampling error. Previous work suggests that autofluorescence imaging (AFI) in combination with a molecular panel might reduce the number of biopsies, but this was not sufficiently sensitive for low-grade dysplasia, now a point for endoscopic intervention. Here we used AFI to direct narrow-field imaging tools for real-time optical assessment of dysplasia and biopsies for a biomarker panel. We compared the new diagnostic algorithm with the current standard. METHODS: A total of 55 patients with BE were recruited at a single tertiary referral center. Patients underwent high-resolution endoscopy followed by AFI. AFI-targeted areas (n=194) were examined in turn by narrow-band imaging with magnification (NBIz) and probe-based confocal laser endomicroscopy (pCLE). Biopsies were taken from AFI-targeted areas and tested using an established molecular panel comprising aneuploidy plus cyclin A and p53 immunohistochemistry. RESULTS: In the per-patient analysis the overall sensitivity and specificity of AFI-targeted pCLE were 100% and 53.6% for high-grade dysplasia/intramucosal cancer and 96.4% and 74.1% for any grade of dysplasia, respectively. NBIz had equal specificity for dysplasia detection (74.1%), but significantly lower sensitivity (57.1%) than pCLE. The time required to perform AFI-targeted pCLE was shorter that that taken by the Seattle protocol (P=0.0004). We found enrichment of molecular abnormalities in areas with optical dysplasia by pCLE (P<0.001), regardless of histologic dysplasia. The addition of the 3-biomarker panel reduced the false positive rate of pCLE by 50%, leading to sensitivity and specificity for any grade of dysplasia of 89.2% and 88.9%, respectively. CONCLUSIONS: The combination of pCLE on AFI-targeted areas and a 3-biomarker panel identifies patients with dysplasia.

A standardized method for quantifying proliferation by Ki-67 and cyclin A immunohistochemistry in breast cancer.

Immunohistochemical analysis of proliferation markers such as Ki-67 and cyclin A is widely used in clinical evaluation as a prognostic factor in breast cancer. The proliferation status of tumors is guiding the decision of whether or not a patient should be treated with chemotherapy because low-proliferative tumors are less sensitive by such treatment. However, the lack of optimal cutoff points and selection of tumor areas hamper its use in clinical practice. This study was performed to compare the Ki-67 and cyclin A expression counted in hot-spot vs average counting based on 5 to 14 random tumor areas in 613 breast carcinomas. We correlated the findings with 10-year follow-up in order to standardize the evaluation of proliferation markers in clinical practice. A significant correlation was found between the percentage of positive cells estimated by Ki-67 and cyclin A both by hot-spot and by average counting. Both methods showed that high expression of Ki-67 and cyclin A is associated with more adverse tumor stage. The cutoff value for Ki-67 for distant metastases was set to 22% and to 15%, using hot-spot and average counting, respectively. For cyclin A, the values were set to 14% and 8% using the respective methods. Survival curves revealed that patients with a high hot-spot proliferation index had a significantly greater risk of shorter tumor-free survival. Our findings suggest that the determination of proliferation markers in breast cancer should be standardized to hot-spot counting and that specific cutoff values for proliferation could be useful as prognostic markers in clinical practice. Moreover, we suggest that expression levels of cyclin A could be used as a complementary marker to estimate the proliferation status in tumors, especially those with "borderline" expression levels of Ki-67, in order to more accurately estimate the proliferations status of the tumors.

Cyclin A correlates with YB1, progression and resistance to chemotherapy in human epithelial ovarian cancer.

BACKGROUND: Cyclin A is a cell-cycle regulatory gene and its overexpression promotes tumor cell growth. Y-Box-binding protein 1 (YB1) is a transcription/translation factor involved in tumor growth, invasion, and drug resistance. We investigated whether an association exists between protein products of these genes in epithelial ovarian cancer (EOC) specimens and clinicopathological parameters, patient response and EOC sensitivity to platinum-based first-line chemotherapy. PATIENTS AND METHODS: Cyclin A and YB1 expression were analyzed by immunohistochemistry in 54 human primary EOC tissues. Immunolabeling of both proteins was graded according to their staining intensity (scale 0-3) and the proportion of immunostained cancer cells (scale 0-4) to obtain a staining index (SI; value=0-12). RESULTS: Significantly higher cyclin A immunostaining (SI≥4) in EOC specimens was discovered in patients with advanced (International Federation of Gynaecology and Obstetrics (FIGO) III and IV, p=0.003), poorly differentiated (G3, p<0.001) tumors, and tumors of those with residual disease>1 cm (p=0.001). YB1 immunostaining was significantly higher in EOCs from patients with suboptimal debulking (p=0.025). Over-expression of cyclin A (SI≥9) in EOCs was significantly linked with poorer patient response (p=0.001) and higher resistance of tumors to platinum-based first-line chemotherapy (p=0.007), while immunolabeling of YB1 in EOCs was not significantly associated with either of these variables (p>0.05). Cyclin A expression was significantly and positively correlated with that of YB1 (R=0.588, p<0.001). CONCLUSION: Increased cyclin A expression in EOC is related to a more aggressive tumor behavior and predicts the response of patients to first-line platinum-based chemotherapy.

Expression of proliferation markers Ki67, cyclin A, geminin and aurora-kinase A in primary breast carcinomas and corresponding distant metastases.

AIMS: To assess the expression of the following cell cycle regulatory proteins in primary metastatic breast carcinomas (MBCs) and on availability in matched distant metastases (DMs): Ki67, cyclin A, geminin and aurora-kinase A (aurkA); and to compare the expression of these markers in early MBC (EMBC) and late MBC separated into groups according to median time point on metastatic event occurred (28 months). METHODS: The expression of the above mentioned markers was analysed in a total of 47 primary MBCs and 59 DMs (out of which 37 were pairs) by immunohistochemistry. Fourteen breast carcinomas with no relapse over a 10-year follow-up period were utilised as control cases (CBC). RESULTS: Among the MBCs, 22 metastasised to the bone, 4 to the lung and 21 to the central nervous system (CNS). Geminin (p<0.001) and Ki67 (p=0.001) were increased in the MBCs while aurkA and cyclin A showed no difference when compared with CBCs. There were no differences between aurkA, cyclin A and geminin expression in MBCs and DMs in general. Expression of Ki67 was, however, elevated (p=0.027) in DMs. In CNS metastases all markers showed elevated expression as compared to MBCs. In bone metastases, geminin was lower (p<0.001) compared with primary MBCs. In the metastases of the lung, the evaluated markers did not show different expression. According to the median follow-up until the metastatic event, Ki67 was found to be significantly elevated in EMBC (p=0.018). CONCLUSIONS: Ki67 index and geminin distinguish a fraction of MBC with worse prognosis, showing increased levels in the latter in comparison to CBC being tumour-free over a 10-year follow-up period. Ki67 could possibly identify a group of MBCs that develop early DMs.

Imaging analysis of cell cycle-dependent degradation of Cdt1 in mammalian cells.

Numerous cell cycle-regulating proteins are controlled by protein degradation. Recent work shows that ubiquitination-dependent proteolysis plays an important role in once-per-cell cycle control of DNA replication. Cdt1 is a licensing factor essential for assembling the pre-replicative complex on replication origins. Cdt1 is present in G1 phase, but after S phase ubiquitin-mediated proteolysis maintains Cdt1 at low levels. This is important to prevent the re-replication of chromosomal DNA. The cell cycle-dependent degradation of Cdt1 can be monitored by dual staining of the cell nuclei with antibodies against Cdt1- and S/G2-phase marker proteins, such as cyclin A or geminin.

Cucurbitacin-I, a natural cell-permeable triterpenoid isolated from Cucurbitaceae, exerts potent anticancer effect in colon cancer.

Cucurbitacin-I is a triterpenoids found in medicinal plants and have diverse pharmacological and biological activities. In this study, the antitumor effects of cucurbitacin-I on colon cancer and possible roles in apoptosis and cell cycle arrest were investigated. Treatment of SW480 cells, a human colon cancer cells, with cucurbitacin-I decreased cell viability and cell proliferation in a concentration-dependent manner. Also, cucurbitacin-I induced G2/M phase cell cycle arrest in SW480 cells with a decreased expression of cell cycle proteins including cyclin B1, cyclin A, CDK1, and CDC25C. Moreover, cucurbitacin-I induced increased cleavage of caspase-3, -7, -8, -9, and poly ADP ribose polymerase. When we examined the inhibitory effect of cucurbitacin-I on tumor growth in vivo, cucurbitacin-I effectively inhibited the tumorigenicity and growth of CT-26 cells in syngenic BALB/c mice. In summary, the present study showed that cucurbitacin-I reduced colon cancer cell proliferation by enhancing apoptosis and causing cell cycle arrest at the G2/M phase.

High expression of cyclin A is associated with poor prognosis in endometrial endometrioid adenocarcinoma.

Cyclins are a group of proteins that act as activators to cyclin-dependent kinases and are required for normal cell cycle transitions. Cyclin A is involved in the transitions between G1 to S and G2 to M. Its deregulation has been linked to a number of neoplasms, including endometrial cancer. The prognostic significance of cyclin A expression seems to be cancer-specific, and current knowledge on its impact on survival of endometrial cancer is limited. This study aimed to investigate the effect of cyclin A expression on cancer-specific survival and its correlation with conventional prognostic factors in endometrioid adenocarcinoma. Biopsies obtained from 211 patients were immunohistochemically stained for cyclin A and differences in expression analyzed at the Oulu University Hospital. Patients were divided into two groups utilizing the ROC curve. Further survival analyses were carried out between these two groups. In this study, we show that cyclin A expression correlates with tumor grade and FIGO stage. We also show that cyclin A is an independent prognostic factor in endometrioid adenocarcinoma. Whether cyclin A plays a role in tumorigenesis or merely is a marker of increased proliferation requires further studies.

The impact of cell proliferation markers and p53 mutation status on prognosis of non-metastatic colon cancer.

BACKGROUND AND OBJECTIVES: We aimed to evaluate the prognostic value of cell cycle proteins and p53 together with clinicopathologic features in non-metastatic resected colon cancer. METHODS: One hundred nine patients who were diagnosed with resected colon cancer between 2006 and 2011 were analyzed retrospectively. Immunohistochemical staining analyses were used to evaluate the expression of cyclins D1 and A, p53 and Ki-67 in tumor tissue. RESULTS: High cyclin D1 and cyclin A expression was more common in stage II than stage III tumors. Disease recurrence was more frequent in tumors with low cyclin D1 expression (P = 0.05). No significant association was observed between p53, Ki-67 or cyclin A expression and the risk of relapse and/or death. Multivariate analysis showed that the strongest predictor for a shorter disease-free survival period was extracapsular nodal invasion (ECNI). CONCLUSIONS: We were not able to establish a strong association between patient prognosis and cyclins D1 and A, p53 or Ki-67 expression. However, a negative correlation between cyclin D1 and cyclin A expression and disease stage as well as more frequent relapses in patients with low expression of cyclin D1 suggested that cyclins may be predictive for early relapse in non-metastatic colon cancer.

Overexpression of cyclin A in oral dysplasia: An international comparison and literature review.

Oral squamous cell carcinoma (OSCC) is one of the most debilitating cancers in the world and while its causes have been heavily researched, the outcome remains grim. Most of these cancers are identified in the late stage and as a result treatment options are limited. Therefore, researchers have focused their efforts on recognizing and identifying dysplastic tissue that has an increased chance of progressing to cancer. Research has begun to look at cell cycle dysfunctions and in particular, aberrant protein functions as a way of identifying the cellular mechanism at fault. The overexpression of a group of regulatory proteins called cyclins has been demonstrated in many types of dysplasia and carcinomas. Although researchers have identified several different types of cyclins as potential culprits, we chose to focus our study primarily on the overexpression of cyclin A. While most research on oral dysplasia and OSCC has been focused on cyclin D, studies have been done on cyclin A. While the etiology of oral dysplasia/SCC appears to be multifactorial, we chose to compare our results with those of similar studies performed across the globe. The social factors, such as the increased use of tobacco that may have contributed to our results, were compared with similar studies performed in Europe and Asia. While our results were remarkably similar and demonstrated a link between the overexpression of cyclin A in oral dysplasia, there exists some differences and thus may require a multicenter, longitudinal study.

Real time detection of cell cycle regulator cyclin A on living tumor cells with europium emission.

Six water-soluble europium complexes (Eu-L1-P(n) and Eu-L2-P(n), n = 1, 2 and 3) with one antenna chromophore, two different linkers (L1 and L2) and three proposed cyclin A specific peptides (P1: -GAKRRLIF-NH2; P2: -GGAKRRLIF-NH2; P3: -Hex- GAKRRLIF-NH2) have been synthesized. With structural information available, comparisons of the cyclin grooves of cyclin A and the six europium complexes have been made, and insights have been gained into the determinants for peptide binding and the foundation of differential binding. Experiment-wise, the linear and two-photon induced photophysical properties of these conjugates were monitored in aqueous solution. Numerous in situ/in vitro biological assays have been carried out, such as responsive emission changes in situ/in vitro, Western blot and cellular uptake. As imaging agents, complexes with peptides P3: -Hex-GAKRRLIF-NH2 showed high selectivity to cyclin A in numerous cancer cells. When it comes to responsive optical signal changes, complex Eu-L2-P3 exhibited a threefold emission enhancement upon binding with cyclin A (100 nM cyclin A, ϕ = 8% to 21%, log KB = 5.83, detection limit = 5 nM), and this could be initiated by the shortened distance between the antenna and the lanthanide after they bind/get into cyclin A. It is promising that our compounds (especially compound Eu-L2-P3) could serve as the template for structure-guided efforts to develop potential imaging therapeutics on the basis of selective imaging of CDK2/cyclin A activity.

Markers of cell division cycle in glioblastoma: significance in prediction of treatment response and patient prognosis.

OBJECTIVE: To investigate whether expression of regulatory components of the cell division cycle can be used independently to predict survival and response to adjuvant therapy in glioblastomas. METHOD: A tissue micro-array, constructed using glioblastomas (n = 66), was stained using antibodies against minichromosome maintenance protein-2 (Mcm-2), expressed throughout the cell-division cycle; geminin, a protein that prevents re-initiation of DNA replication; and cyclin A, an S-phase cyclin. A semi-quantitative labelling index (LI) was calculated using an average of 18 high-power fields (hpf) in three replicate cores. The patients were divided into two groups: Group 1 (n = 50) underwent surgery and radiotherapy with 24 patients receiving temozolomide, and Group 2 (n = 16) received surgical treatment only. RESULTS: The LIs (median +/- IQR) for Group 1 were as follows: Mcm-2, 36.7% (22.9%-51.8%); geminin, 7.8% (5.8%-10.5%); and cyclin A, 4.2% (2.4%-6.9%). Elevated LIs, higher than the median, for geminin and cyclin A correlated with prolonged survival when the tumours received adjuvant therapy (Kaplan-Meier curves, p = 0.0046 and p = 0.0063 for geminin and cyclin A, respectively). Linear regression analysis revealed positive correlations with survival for Mcm-2 (p = 0.0376), geminin (p = 0.0006) and cyclin A (p = 0.004). In Group 2, there was no relationship between the patient survival and the LI for any marker. CONCLUSIONS: Geminin and cyclin A, each show potential as independent prognostic markers in glioblastomas receiving adjuvant therapy. This may reflect the fact that both geminin and cyclin A estimate proliferating tumour cell subpopulations sensitive to radio/chemotherapy. These markers could provide valuable prognostic information, even in small biopsies, especially if combined with O(6)MGMT expression and 1p;19q deletion status.

High proliferation is associated with inferior outcome in male breast cancer patients.

Assessment of proliferation is important in female breast cancer and individual treatment decisions are based upon its results, especially in the luminal subgroups. Gene expression analyses fail to group male breast cancer into the intrinsic subgroups previously established in female breast cancer. Even though proliferation has been shown to divide male breast cancer into molecular subgroups with different prognoses, the clinical importance of proliferation markers has not yet been elucidated. Previous studies in male breast cancer have demonstrated contradictory results regarding the prognostic impact of histological grade and Ki-67, parameters strongly associated with proliferation. The aim of the present project was to study proliferation in male breast cancer by assessing other proliferation-related markers viz. cyclins A, B, D1 and mitotic count. A total of 197 male breast cancer cases with accessible paraffin-embedded material and outcome data were investigated. Immunohistochemical stainings were performed on tissue microarrays. Kaplan-Meier estimates and the Cox proportional regression models were used for survival analyses with breast cancer death as the event. The subset of patients with high expression of cyclin A (hazard ratio (HR) 3.7; P=0.001) and B (HR 2.7; P=0.02) demonstrated a poorer survival. Furthermore, high mitotic count was associated with an increased risk of breast cancer death (HR 2.5; P=0.01). In contrast, cyclin D1 overexpression was predictive of better breast cancer survival (HR 0.3; P=0.001). In conclusion, high levels of cyclin A and B expression and an elevated mitotic count result in a two to threefold higher risk for breast cancer death, whereas cyclin D1 overexpression halves the risk. The clinical utility of these proliferation markers needs further elucidation.

Expression of cyclin A, B1 and D1 after induction of cell cycle arrest in the Jurkat cell line exposed to doxorubicin.

Jurkat human lymphoblastoid cells were incubated in increasing concentrations of doxorubicin (0.05, 0.1 and 0.15 µM) to induce cell death, and their expression of cyclin A, B1 and D1 was evaluated by flow cytometry (cell cycle progression, Annexin V assay, percentages and levels of each of the cyclins), transmission electron microscopy (ultrastructure) and confocal fluorescence microscopy (expression and intracellular localization of cyclins). After low-dose doxorubicin treatment, Jurkat cells responded mainly by G2/M arrest, which was related to increased cyclin B1, A and D1 levels, a low level of apoptosis and/or mitotic catastrophe. The influence of doxorubicin on levels and/or localization of selected cyclins was confirmed, which may in turn contribute to the G2/M arrest induced by the drug.

Clinical, genetic, and immunohistochemical characterization of 70 Ukrainian adult cases with post-Chornobyl papillary thyroid carcinoma.

BACKGROUND: Increased incidence of papillary thyroid carcinoma (PTC) is observed as a consequence of radiation exposure in connection to the Chornobyl nuclear plant accident in 1986. In this study, we report a cohort of adult Ukrainian patients diagnosed with PTC from 2004 to 2008 following exposure at the age of 18 years or younger. METHODS: In total, 70 patients were identified and clinically characterized. The common BRAF 1799T>A mutation was assessed by pyrosequencing, the RET/PTC1 and RET/PTC3 (NCOA4) rearrangements by RT-PCR, and the expression of Ki-67 (MIB-1 index), BCL2, cyclin A, and cyclin D1 by immunohistochemistry. RESULTS: In total, 46/70 (66%) cases carried a BRAF mutation and/or a RET/PTC rearrangement. A BRAF mutation was detected in 26 tumors, RET/PTC1 in 20 cases, and RET/PTC3 in four cases. In four of these cases, BRAF mutation and RET/PTC rearrangement were coexisting. The BRAF mutation was underrepresented among PTCs with accompanying chronic lymphocytic thyroiditis (CLT) compared with PTCs without this feature (12 vs 44%). MIB-1 proliferation index determined by double staining with leukocyte common antigen was low (mean 0.8%; range 0.05-4.5%). Moreover, increased expression of cyclin A was observed in PTCs with a tumor size >2 cm compared with PTCs ≤2 cm (1.2 vs 0.6%). BCL2 and cyclin D1 showed frequent expression but without associations to clinical characteristics or amplification of the CCND1 locus. CONCLUSIONS: Our results suggest that this cohort has frequent BRAF mutation, RET/PTC1 rearrangement, and low proliferation index. Furthermore, BRAF 1799T>A was underrepresented in PTCs with CLT, and cyclin A expression was associated with increased PTC tumor size.

Cyclin A and cyclin B1 overexpression in differentiated thyroid carcinoma.

Approximately 30% of patients with thyroid nodules have indeterminate or suspicious fine-needle aspiration (FNA) biopsy results. These patients usually undergo thyroidectomy because of cancer risk. Our aim was to determine diagnostic value of cyclin A and cyclin B1 immunohistochemistry added to routine cytology and their expression on histological sections. We studied the expression of cyclin A and cyclin B1 in FNA biopsies and resection specimens of 168 indeterminate or suspicious FNA biopsy results retrospectively at an academic hospital using immunohistochemistry. Malignant histopathology consisted 64 of resection specimens (58 papillary, 4 follicular, 1 medullary, and 1 Hürthle cell carcinoma). Cyclin A was overexpressed in 51.5% of malignant cases in contrast to 31.7% of 104 benign pathology specimens (P = 0.025). Cyclin B1 was positive in 39.1% of malignant specimens in contrast to 15.4% of benign cases (P = 0.001). Cyclin A overexpression was not linked to cyclin B1 overexpression. No association was found between overexpression of cyclin A, cyclin B1 and age, thyroiditis, multifocality, tumor size, extra-thyroidal extension, capsule infiltration, lymph node and distant organ metastases and TNM stage in malignant cases. Female patients with thyroid carcinoma overexpressed significantly more cyclin B1 than male patients (P = 0.015). Retrospective analysis of cyclin A and cyclin B1 in FNA biopsies yielded negative results for both benign and malignant cases. In conclusion, cyclin A and cyclin B1 are useful markers in the distinction of benign and malignant thyroid tumors and can increase diagnostic accuracy.