Distinct Dynamics of Mitotic Transition in B-Cell Lymphoma and Reactive B-Cell Lymphoproliferations Determined by H3S10 Phosphohistone Immunolabeling.

OBJECTIVES: Clonal selection in the follicular germinal centers in lymphatic tissues is accompanied by an intense proliferation of polyclonal B cells in a precisely regulated fashion. In contrast, B-cell neoplasias proliferate autonomously due to endogenous stimuli. The cell kinetic activity is obvious at many levels including progressive chromatin modification and elevated mitotic rates. We asked if there are differences in the kinetics of histone H3S10 phosphorylation required for mitotic entry between highly proliferating B cells of reactive germinal centers and in B-cell lymphomas with different proliferative capacity. MATERIAL AND METHODS: Phospho-H3 histone (pH3S10)-specific immunohistochemistry was applied to cultivated cell, reactive and selected indolent and aggressive lymphoma samples (diffuse large B-cell lymphoma, Burkitt lymphoma, lymphoblastic lymphoma, follicular lymphoma and small lymphocytic lymphoma). Microscopic quantification of the "dot-type" (representing late G2 phase) and "mitotic" immunolabeling patterns per field of view was performed and compared with classical cell proliferation markers. RESULTS: In addition to the dense homogeneous chromatin labeling highlighting mitotic figures, we stated a selective dot-type nuclear labeling representing ongoing chromatin condensation in premitotic G2 phase cells. While cell proliferation and mitotic counts correlated in general with histology, statistical analysis indicated an accumulation of G2 phase pH3S10 pattern in the reactive germinal centers in contrast to lymphomas. The dot-type G2 staining pattern was surprisingly overrepresented (1,321.7 ± 356.5/10 HPF) in the reactive germinal centers compared to aggressive lymphomas (101.3 ± 33.1) (p < 0.005). The relative G2/M value was significantly higher (4.6 ± 0.6) in reactive germinal center B cells than in any lymphoma entity evaluated (0.7 ± 0.2 in Burkitt lymphoma, 0.9 ± 0.4 in grade 3b follicular lymphoma, 1.3 ± 1.1 in diffuse large B-cell lymphoma, 1.5 ± 0.6 in lymphoblastic lymphoma, and 0.9 ± 0.2 in small lymphocytic lymphoma). CONCLUSIONS: pH3S10 immunohistochemistry enabled the presentation of significant differences in the cell cycle kinetics between reactive and neoplastic B-cell lymphoproliferations. Accumulation of G2 phase B cells in reactive folliculi directs to physiological G2/M checkpoint blockade. In contrast, accelerated G2/M transition in lymphomas is potentially associated with impaired genomic repair and cell death mechanisms.

[Biologic mechanisms of mitotic abnormalities and chromosome number changes in malignant tumors]. / A sejtosztódás rendellenességeivel és a kromoszómaszám változásával kapcsolatos biológiai mechanizmusok vizsgálata malignus daganatokban.

The main goal of this work was to study the effect of Aurora kinase expression on cell ploidy and tumorigenesis. Fifty invasive breast cancer, 50 diffuse large B-cell lymphoma and 10 reactive lymph node samples were recruited in the study. Because of the significant correlation with the overall cell proliferation rate, the overexpression of Aurora B could not be stated on the basis of kinase expressing tumor cell fractions alone. The relative expression of Aurora B kinase is better reflected by the AMI index which represents the Aurora B expression in relation to the whole proliferative fraction of the tumor. A higher relative Aurora B expression was associated with higher mitotic activity in B-cell lymphoma. FISH analysis of the AURKB locus did not show any gains or amplifications in the samples analyzed. On the other hand, we have observed the loss of the gene in breast carcinoma and lymphoma samples as well. A strong correlation was shown between AURKB and TP53 copy numbers: AURKB loss was associated with TP53 deletion in all samples. According to our results on breast carcinoma, losses at 17p13.1 and chromosome 17 aneusomy determined by FISH showed a statistically significant correlation. Our study presents the frequent occurrence of chromosome 17 aneusomy in breast carcinoma and B-cell lymphoma samples. Chromosome 17 aneusomy evaluated by FISH correlated with aneuploidy determined by flow cytometry. Direct correlation between kinase expression and ploidy could not be shown. The highest AMI values were seen in B-ALCL samples, and it was associated with high chromosome 17 copy numbers and mitotic activity. The damaged Aurora B kinase function results in regulatory deficiencies in the CPC complex leading to mitotic errors, while p53 deficiency helps malignant cells to survive due to insufficient activation of the intrinsic apoptotic pathways. The upregulation of Aurora kinase B function may cause error in an important mitotic checkpoint, thus resulting in induction of aneuploid cell populations. These parallel effects finally increase the complexity of mitotic abnormalities and generate aneuploid cell populations.

[Occurrence and molecular pathology of high grade gliomas]. / Magas grádusú gliomák elofordulása és molekuláris patológiája.

BACKGROUND: Glial tumours represent the most frequent type of primary brain cancers. Gliomas are characterized by heterogeneity that makes the diagnosis, histological classification and the choosing of correct therapy more difficult. Despite the advances in developing therapeutic strategies patients with malignant gliomas have a poor prognosis; therefore glial tumours represent one of the most important areas of cancer research. There are no detailed data on the epidemiology of gliomas in Hungary. METHODS: In the first section of our publication, we analysed the histological diagnosed cases between 2007 and 2011 at the Institute of Pathology, University of Debrecen Medical and Health Science Centre. We analyzed the incidence of 214 high-grade gliomas by tumor grades, gender, age, and the anatomical localization. RESULTS: The majority of cases were glioblastoma (182 cases), and the remaining 32 cases were anaplastic gliomas. The mean age of patients was 57 years (+/- 16.4), and the male:female ratio was 1.1:1. The most frequent area of tumors was the frontal lobe followed by the temporal, parietal and occipital lobe. We include new findings published recently about glioma pathogenesis, molecular pathways, mutant genes and chromosomal regions. We explain briefly the role of selected important genes in glioma genesis and give an update on knowledge provided by modern molecular methods, which could beneficially influence future therapy and the diagnosis of gliomas.

Aurora kinase B expression in breast carcinoma: cell kinetic and genetic aspects.

BACKGROUND: Mitotic deregulations may contribute significantly to cell division errors and the development of aggressive tumor cells. The mitotic kinase Aurora B is essential for chromosome segregation. Its gene is located at 17p13 in close proximity to the TP53 gene. Although the frequent alteration of this locus is well known, the information about the AURKB status and protein expression is limited. METHODS: 50 breast carcinoma cases were evaluated for 17p13 status and chromosome 17 ploidy by FISH and for Aurora B protein by immunohistochemistry. RESULTS: Aurora B protein expression showed a strong correlation with cell proliferation (regression coefficient = 0.77). Therefore, the Aurora B/MIB-1 index was used as a measure of expression, which showed a wide range (1-35%, mean 0.32, SD ± 0.28). A gain in the 17p13 chromosome locus could not be shown while a deletion was stated in 10/50 cases including a subset with TP53 and AURKB codeletion in 6/10 cases. The loss of TP53/AURKB loci strongly correlated with aneusomy (p < 0.0001). CONCLUSION: Elevated Aurora B expression frequently occurs due to an increased cell proliferation rate in breast carcinoma. Codeletion of TP53 and AURKB at 17p13 indicates a concerted mechanism leading to the survival of cell clones with deficient mitotic kinase function which could contribute to the formation of aneuploid cells and an aggressive tumor phenotype.

Mitotic Index Determined by Phosphohistone H3 Immunohistochemistry for Precise Grading in Follicular Lymphoma.

The World Health Organization classification recommends follicular lymphoma (FL) grading (G1-3) by considering centroblast number, while also suggesting its influence on disease outcome. As centroblast counting and other proliferation markers have limitations, we looked for more specific measures of cellular activity in FL. Phosphorylated histone H3 (pHH3) was widely applied for the objective detection of mitotic activity in different tumors. The aim was to evaluate the utility of pHH3 protein in FL grading and compare its value with the classical features of cell proliferation. Representative samples from 48 FL patients and 9 samples with follicular hyperplasia were examined. Hematoxylin-eosin-based mitosis index (HE-MI), number of mitotic figures based on anti-pHH3 immunohistochemical staining (pHH3-MI), and percentage of Ki-67-positive cells [proliferation index (PI)] were determined and compared with centroblast-based histologic grade. PHH3-MI showed significant correlation with HE-MI (r=0.85, P<0.0001) and PI (r=0.84, P<0.0001). All 3 cell proliferation parameters showed significant correlation with histologic grade: HE-MI versus grade, r=0.85 (P<0.0001); PI versus grade, r=0.74 (P<0.0001); pHH3-MI versus grade, r=0.80 (P<0.0001). PHH3-MI showed continuous increase with the histologic grade. The pHH3-MI value was distinctive between the G2 and the G1 FL groups (P<0.0001) and was increased in G3 FL compared with that in the G2 FL group (P=0.0020). In conclusion, easy-to-perform mitotic counting following phosphohistone H3 immunohistochemistry (pHH3-MI) correlates well with centroblast-based grading. PHH3 immunohistochemistry offers a reliable quantification tool supporting lymphoma grading and can be recommended as an additional parameter for the precise subcategorization of FL cases.

Genome wide analysis of TLR1/2- and TLR4-activated SZ95 sebocytes reveals a complex immune-competence and identifies serum amyloid A as a marker for activated sebaceous glands.

Toll-like receptors (TLR) 2 and 4 are active in sebaceous glands and play a central role in the development of acne. Still, there is only limited knowledge on their effect on sebocytes. In this work we performed global gene expression profile analysis with functional clustering of the differentially regulated genes of TLR1/2 (PAM3CSK4)- and TLR4 (lipopolysaccharide [LPS])-activated SZ95 sebocytes. Both TLR1/2- and 4-activation promoted inflammation in a similar manner already at an early time-point (6 hours), regulating genes involved in inflammation, wound healing and chemotaxis reflecting a more complex cytokine and chemokine regulation than previously known. Importantly, lipid metabolism, the primary feature of sebocytes, was affected at the level of gene expression only at a later time point (24 hours) indicating that sebocytes prioritize to exert a pro-inflammatory phenotype when confronted with a danger signal. Supporting the biological relevance of our results, a meta-analysis revealed that the genes showing the strongest up-regulation were also found up-regulated in acne. Of these genes, serum amyloid A 1/2 (SAA1/2) was confirmed to be a suitable protein marker for in vivo activated sebocytes, underlining their immune-competence, which is structurally defined within sebaceous glands of acne and rosacea skin samples. Altogether our findings demonstrate that sebocytes are not only positioned at the end point of inflammation but are actively involved in shaping the inflammatory response with putative diagnostic and therapeutic relevance.

Down-regulation of increased TRAF6 expression in the peripheral mononuclear cells of patients with primary Sjögren's syndrome by an EBV-EBER1-specific synthetic single-stranded complementary DNA molecule.

AIM: We described earlier a simultaneously increased that the increased expression of miRNA-146a/b was accompanied by an increase in the expression of and TRAF6 and a decrease in the expression of IRAK1 genes in the peripheral mononuclear cells (PBMCs) of patients with primary Sjogren's syndrome (pSS) patients. Recently, the expression of EBV encoded. RNA (EBER) was published in the B cells of salivary glands of in pSS. In the present study, we applied an EBV-EBER1 specific synthetic single stranded complementary DNA molecule (EBV-EBER1-cDNA) to test whether any EBER1 related effect exists also in PBMCs of pSS patients. METHODS: In the PBMCs of pSS patients and healthy controls, we investigated in vitro the effects of a synthetic single stranded EBV-EBER1-cDNA molecule, synthetic double-stranded (ds)RNA polyinosinic-polycytidylic acid [poly (I:C)] and polyadenylic acid potassium salt poly-adenylic acid [poly-(A)] on the expression of TRAF6 gene tested by qRTPCR. The release of interferon -α was detected by ELISA. RESULTS: EBV-EBER1-cDNA resulted in a significant reduction in the expression of TRAF6 in the cells of patients, but in the healthy controls not, whereas the treatments with poly (I:C) and poly-(A) could not reduce the TRAF6 over-expression. No release of EBER1 could be observed in the culture supernatants of patients with pSS. Only the treatment with poly (I:C) resulted in a significant increase of interferon -α release, and only in the heathy controls. No release of EBER1 molecules took place during the culturing of cells. EBV-EBER- cDNA acted functionally on the cells of patients only. CONCLUSION: These findings give a further evidence of the linkage between EBV and pSS, furthermore, they show the possible role of EBV-EBER1 in the induction of increased TRAF6 expression in the peripheral B cells of Sjögren's patients.

Mutant KRAS Status Is Associated with Increased KRAS Copy Number Imbalance: a Potential Mechanism of Molecular Heterogeneity.

Mutation rates determined by allele-specific PCR can be highly different in KRAS exon 2 mutant colorectal carcinoma (CRC) samples suggesting intratumoural heterogeneity. To address the effect of KRAS gene copy number on the relative mutant allele frequency the KRAS locus was individually quantified following FISH analysis in 36 cases. We observed, that mutant KRAS status was associated with an elevated KRAS locus number (2.36 ± 0.42 vs 2.63 ± 0.75; p = 0.037) reflecting an increased aneuploidy status but no true amplification of the locus. In parallel, KRAS locus copy numbers showed significant intercellular variability (1-16 copies/cell nucleus) within individual tumours also indicating to a dynamic intratumoural oscillation of the mutant allele copy number. In conclusion, aneusomy is a common feature of KRAS mutant CRC and KRAS copy number variations may have an impact on the relative mutant allele frequency detected by allele specific PCR/sequencing), potentially leading to subclonal diversity and influencing tumour behaviour.

One-day FISH approach for the high-speed determination of HER2 gene copy status in breast carcinoma.

Fluorescence in situ hybridization (FISH) is a commonly used method to detect chromosomal aberrations, for example, to assess human epidermal growth factor receptor 2 (HER2) gene status in breast carcinoma. The classical FISH approach requires overnight incubation for proper hybridization result. Tissue morphologic features are varying because of aggressive pretreatment and application of high temperatures. To eliminate some of the methodological problems, a new 1-day FISH method was recently introduced. The aim of our study was to evaluate the utility of the Instant Quality FISH with the conventional FISH kit from the same provider (Dako pharmDx) for determination of HER2 status. We performed in situ hybridization on the same 40 invasive breast carcinoma samples with both probe kits, and HER2/CEN17 and chromosome 17/cell nucleus ratios were calculated. FISH signal stability was also tested by the reassessment of the slides after 2 months storage. The accordance regarding HER2 gene amplification status between the 2 FISH kits tested was 100%. There was an excellent correlation between HER2/CEN17 ratios with a concordance correlation coefficient of 0.958 and correlation coefficient (R) of 0.959. The 1-day HER2 Instant Quality FISH diagnostic kit points with fast and stable reaction showing the same result in the diagnostic practice when compared with the conventional overnight FISH method.

Mitotic failures in cancer: Aurora B kinase and its potential role in the development of aneuploidy.

One of the basic requirements during the process of cell division is to maintain genetic integrity and ensure normal ploidy. The family of Aurora kinases, composed of Aurora A, B and C, takes a major role in the control of centrosome cycle, mitotic entry, chromosome condensation and coordination of chromosomal movements. Deregulation of kinase expression was described in a series of different malignancies which was also associated with aneuploidy. Recently, Aurora kinases gained significant interest as potential therapeutic targets in oncology. While there is increasing evidence about the activities of Aurora A kinase during cancer progression, data are controversial regarding the role of Aurora B. In this review the biology of Aurora kinases and its potential relation to cancer progression is discussed with special focus on functional changes and determination of Aurora B kinase.

Primary uterine NK-cell lymphoma, nasal-type: a unique malignancy of a prominent cell type of the endometrium.

Natural killer (NK) cells host in the human endometrium with dedicated role in reproductive physiology. Interestingly, malignant transformation of these specialized cells has not been presented thus far. Here we report a primary endometrial NK-cell lymphoma of a 48 year-old patient presenting with irregular bleeding. The endometrial curetting showed a dense lymphomatous infiltrate demonstrating highly infiltrative aggressive features with characteristic angiocentric, partially angiodestructive growth pattern and accompanying focal necroses. The lymphoma cells displayed a CD3ε/CD56/TIA-1/granzyme-B-positive and CD5/CD4/CD8/TCRγδ-negative immunophenotype, proved to be positive for Epstein-Barr virus by EBER in situ hybridization, and revealed no clonal T-cell receptor gene rearrangement. The diagnosis of uterine extranodal NK-cell lymphoma, nasal-type was made. Clinically, the disease was limited to the uterus at diagnosis, but progressed rapidly, and the patient died within 5 months due disseminated lymphoma, irrespective of intensive chemotherapy. Genuine NK-cell lymphomas occurring in the uterus as primary site seem to be rare making the therapeutic decisions extremely complicated.