Clonality testing of lymphoproliferative disorders in a large cohort of primary and consultant biopsies.

BACKGROUND: Lymphoproliferative disease often presents the clinician and pathologist with a diagnostic dilemma, particularly in the early course of the disease. METHODS: We used modified BIOMED-2 protocols to detect monoclonal expansions of immunoglobulin heavy chain (IgH) and T-cell receptor (TCR) genes in 957 formalin-fixed paraffin-embedded samples from 717 patients. To eliminate false-positive results, heteroduplex analysis was used after PCR reactions. The impact of different fixatives on DNA quality and performance of PCR was assessed. RESULTS: In the class of B lymphomas we detected clonal IgH rearrangement in nearly 80% of cases and in the class of T lymphomas in 64% of cases. Performance of the assays was 94.7% and 92.5% for IgH and TCR clonality, respectively. Clonality rates in various B and T lymphomas were in concordance with previous studies. We also present 10 difficult cases where PCR analysis of IgH and TCR gene rearrangements significantly contributed to a decision on the correct diagnosis. CONCLUSION: These results confirm that the PCR-based analysis is suitable as a routine method and is helpful in establishing a diagnosis in morphologically unclear cases.

Clinicopathological correlations of the immunoprofile in diffuse large B-cell lymphoma NOS - a single institutions experience.

The experimental platform in hematooncology is still searching for more valid prognostic and predictive factors on clinical, morphological and molecular levels. The bridge closer to daily practice is so-called translation medicine and from this point of view we have tried to sort diffuse large B-cell lymphoma not otherwise specified. The applied methodological approaches are morphology, indirect immunohistochemistry on formaline-fixed, parrafin-embeded tissue, Hans classifier sorting, expression of Bcl-2, CD5, CD20, CD30 and NfκB proteins in comparison with the clinical (Ann Arbor stage, IPI, aa-IPI, PFS, OS), laboratory and cytogenetic results (complex and simplex karyotypes). Statistical analysis included Cox regressive analysis, Mann-Whitney and Kruska-Wallis test. The interval of PFS and OS has been assessed according to Kaplan-Meier analysis. According to Hans classifier 11 cases (18.7 %) could not be sorted exactly into GCB/nonGCB- like subgroups. All relapsing cases bear negative expression of CD10 and 28 cases of non-relapsing cases showed positive expression of CD10. The "third" - GCB-like/nonGCB-like unsortable subgroups shared a very similar course of PFS with the nonGCB-like subgroup and a worse clinical course of OS. Statistically nonsignificantly better response to chemotherapy was shown by cases with positive Bcl-2 expression of more than 30 %. Statistically nonsignificantly better OS and PFS was shown by cases with a proliferation index Ki67 more than 70 %. The study detected 17 cases (28.8 %) with a nuclear expression of p50 and one case with nuclear expression of p65 (1.7 %) which may imply the possibility of NfκB signaling pathway activation. A statistically nonsignificant realationship of p50 expression and OS/PFS was indicated.

Mechanisms of natural brassinosteroid-induced apoptosis of prostate cancer cells.

Brassinosteroids (BRs) are a group of polyhydroxylated sterol derivatives with important regulatory roles in various plant physiological processes. The aim of this study was to examine the mechanism of the antiproliferative activity of natural BRs 28-homocastasterone (28-homoCS) and 24-epibrassinolide (24-epiBL) in hormone-sensitive and -insensitive (LNCaP and DU-145, respectively) human prostate cancer cell lines. The effects of BRs on prostate cancer cells were surveyed using flow cytometry, Western blotting, TUNEL, DNA ladder assays and immunofluorescence analyses. The studied BRs inhibited cell growth and induced G(1) blocks in LNCaP cells accompanied by reductions in cyclin D(1), CDK4/6 and pRb expression. Following BR treatment of DU-145 cells, increases in proportions of cells in the G(2)/M phase of cell cycle were observed, accompanied by down-regulation of cyclins A and B(1). Changes in AR localization patterns in LNCaP cells treated with BRs were shown by immunofluorescence analysis. Furthermore, apoptotic detection methods demonstrated induction of apoptosis mediated by BRs in both cell lines, although changes in the expression of apoptosis-related proteins were modulated differently by 28-homoCS and 24-piBL in each cell line. The studied BRs seem to exert potent growth inhibitory and pro-apoptotic effects and could be therefore highly valuable new candidates for prostate anticancer drugs.

Tumour suppressor PTEN regulates cell cycle and protein kinase B/Akt pathway in breast cancer cells.

BACKGROUND: PTEN is a tumour suppressor protein with phosphatase activity frequently altered in several types of human cancers. MATERIALS AND METHODS: The PTEN effect was studied on the cell cycle (by bromdeoxyuridine incorporation) and on the phosphatidylinositol-3-kinase/protein kinase B/Akt (PI3-K/PKB/Akt) pathway regulating proteins (by immunocytochemical, Western blot analysis and kinase assay) upon transfection of wild-type PTEN and its mutant H123Y in breast cancer cell lines. RESULTS: The expression of the important proteins in the MCF-7 and BT-549 cells was characterised and the cellular localisation of PTEN was analysed. Transfection of H123Y led to the down-regulation of p27(Kip1) and p21(Waf1/Cip1) protein levels and the up-regulation of phosphorylated PKB/Akt. An overexpression of PTEN decreased cyclin E/cdk2 activity and inhibited S-phase entry in MCF-7. In BT-549 these changes were not observed, but overexpression of PTEN led to a diminution of PKB/Akt phosphorylation. CONCLUSION: PTEN function is mediated through the inhibition of the cell cycle and PKB/Akt phosphorylation in breast cancer cells.

Microarray analysis of bicalutamide action on telomerase activity, p53 pathway and viability of prostate carcinoma cell lines.

Bicalutamide is a non-steroidal anti-androgen commonly used in the treatment of prostate carcinoma. We analysed the transcriptional response to bicalutamide treatment with the aim of explaining the inhibition of telomerase in the androgen-sensitive cell line LNCaP and the effects of bicalutamide on the androgen-insensitive cell line DU145. Cells treated with 80 muM bicalutamide in steroid-depleted medium for 1 day were analysed in duplicate by Affymetrix Human Genome Focus Arrays. Response to bicalutamide in LNCaP cells was represented by downregulation of androgen-regulated genes, activation of the p53 pathway and inhibition of telomerase, which was associated with downregulation of v-myc avian myelocytomatosis viral oncogene homologue (MYC) and telomerase reverse transcriptase subunit. In DU145 cells we observed the influence of cell density on bicalutamide effectivity such that highly confluent cells showed lesser sensitivity than low confluent ones. In conclusion, we provide an explanation for telomerase inhibition after androgen receptor blockade in LNCaP cells and we also report activation of the p53 pathway in LNCaP cells and in-vitro sensitivity to bicalutamide of low confluent androgen-insensitive DU145 cells. These findings might have implications for both experimental and clinical research into prostate cancer. In particular, activation of the p53 pathway after treatment with 80 microM bicalutamide could justify usage of bicalutamide dosages higher than 150 mg daily in androgen-sensitive carcinoma therapy.