Comparative Analysis of Transcriptomic Changes including mRNA and microRNA Expression Induced by the Xenoestrogens Zearalenone and Bisphenol A in Human Ovarian Cells.

Xenoestrogens are natural or synthetic compounds that mimic the effect of endogenous estrogens and might cause cancer. We aimed to compare the global transcriptomic response to zearalenone (ZEA; mycotoxin) and bisphenol A (BPA; plastic additive) with the effect of physiological estradiol (E2) in the PEO1 human ovarian cell line by mRNA and microRNA sequencing. Estrogen exposure induced remarkable transcriptomic changes: 308, 288 and 63 genes were upregulated (log2FC > 1); 292, 260 and 45 genes were downregulated (log2FC < -1) in response to E2 (10 nM), ZEA (10 nM) and BPA (100 nM), respectively. Furthermore, the expression of 13, 11 and 10 miRNAs changed significantly (log2FC > 1, or log2FC < -1) after exposure to E2, ZEA and BPA, respectively. Functional enrichment analysis of the significantly differentially expressed genes and miRNAs revealed several pathways related to the regulation of cell proliferation and migration. The effect of E2 and ZEA was highly comparable: 407 genes were coregulated by these molecules. We could identify 83 genes that were regulated by all three treatments that might have a significant role in the estrogen response of ovarian cells. Furthermore, the downregulation of several miRNAs (miR-501-5p, let-7a-2-3p, miR-26a-2-3p, miR-197-5p and miR-582-3p) was confirmed by qPCR, which might support the proliferative effect of estrogens in ovarian cells.

Suppressing the PI3K/AKT Pathway by miR-30d-5p Mimic Sensitizes Ovarian Cancer Cells to Cell Death Induced by High-Dose Estrogen.

MicroRNAs are short non-coding RNA molecules that are involved in tumor development and are considered to be promising candidates in cancer therapy. Here, we studied the role of miR-30s in the pathophysiology of ovarian cancer. According to our results miR-30a-5p, miR-30d-5p, and miR-30e-5p were overexpressed in the estrogen receptor α (ERα)-expressing PEO1 cell line compared to A2780 that lacks this receptor. Furthermore, the expression of miR-30a-5p, miR-30d-5p, and miR-30e-5p were induced in response to high-dose estrogen treatment in PEO1 where intensive cell death was observed according to the induction of apoptosis and autophagy. Lacking or blocking ERα function reduced tolerance to high-dose estrogen that suggests the importance of ERα-mediated estrogen response in the maintenance of proliferation. MiR-30d-5p mimic reduced cell proliferation in both A2780 and PEO1. Furthermore, it decreased the tolerance of PEO1 cells to high-dose estrogen by blocking the ERα-mediated estrogen response. This was accompanied by decreased SOX4 expression that is thought to be involved in the regulation of the PI3K/AKT pathway. Blocking this pathway by AZD8835 led to the same results. MiR-30d-5p or AZD8835 sensitized PEO1 cells to tamoxifen. We suggest that miR-30d-5p might be a promising candidate in the therapy of ovarian cancer.

Circulating Cell-Free Nucleic Acids: Main Characteristics and Clinical Application.

Liquid biopsy recently became a very promising diagnostic method that has several advantages over conventional invasive methods. Liquid biopsy may serve as a source of several important biomarkers including cell-free nucleic acids (cf-NAs). Cf-DNA is widely used in prenatal testing in order to characterize fetal genetic disorders. Analysis of cf-DNA may provide information about the mutation profile of tumor cells, while cell-free non-coding RNAs are promising biomarker candidates in the diagnosis and prognosis of cancer. Many of these markers have the potential to help clinicians in therapy selection and in the follow-up of patients. Thus, cf-NA-based diagnostics represent a new path in personalized medicine. Although several reviews are available in the field, most of them focus on a limited number of cf-NA types. In this review, we give an overview about all known cf-NAs including cf-DNA, cf-mtDNA and cell-free non-coding RNA (miRNA, lncRNA, circRNA, piRNA, YRNA, and vtRNA) by discussing their biogenesis, biological function and potential as biomarker candidates in liquid biopsy. We also outline possible future directions in the field.

The Cell-Free Expression of MiR200 Family Members Correlates with Estrogen Sensitivity in Human Epithelial Ovarian Cells.

Exposure to physiological estrogens or xenoestrogens (e.g., zearalenone or bisphenol A) increases the risk for cancer. However, little information is available on their significance in ovarian cancer. We present a comprehensive study on the effect of estradiol, zearalenone and bisphenol A on the phenotype, mRNA, intracellular and cell-free miRNA expression of human epithelial ovarian cell lines. Estrogens induced a comparable effect on the rate of cell proliferation and migration as well as on the expression of estrogen-responsive genes (GREB1, CA12, DEPTOR, RBBP8) in the estrogen receptor α (ERα)-expressing PEO1 cell line, which was not observable in the absence of this receptor (in A2780 cells). The basal intracellular and cell-free expression of miR200s and miR203a was higher in PEO1, which was accompanied with low ZEB1 and high E-cadherin expression. These miRNAs showed a rapid but intermittent upregulation in response to estrogens that was diminished by an ERα-specific antagonist. The role of ERα in the regulation of the MIR200B-MIR200A-MIR429 locus was further supported by publicly available ChIP-seq data. MiRNA expression of cell lysates correlated well with cell-free miRNA expression. We conclude that cell-free miR200s might be promising biomarkers to assess estrogen sensitivity of ovarian cells.

Circulating miRNA Profiling in Plasma Samples of Ovarian Cancer Patients.

Ovarian cancer is one of the most common cancer types in women characterized by a high mortality rate due to lack of early diagnosis. Circulating miRNAs besides being important regulators of cancer development could be potential biomarkers to aid diagnosis. We performed the circulating miRNA expression analysis in plasma samples obtained from ovarian cancer patients stratified into FIGO I, FIGO III, and FIGO IV stages and from healthy females using the NanoString quantitative assay. Forty-five miRNAs were differentially expressed, out of these 17 miRNAs showed significantly different expression between controls and patients, 28 were expressed only in patients, among them 19 were expressed only in FIGO I patients. Differentially expressed miRNAs were ranked by the network-based analysis to assess their importance. Target genes of the differentially expressed miRNAs were identified then functional annotation of the target genes by the GO and KEGG-based enrichment analysis was carried out. A general and an ovary-specific protein-protein interaction network was constructed from target genes. Results of our network and the functional enrichment analysis suggest that besides HSP90AA1, MYC, SP1, BRCA1, RB1, CFTR, STAT3, E2F1, ERBB2, EZH2, and MET genes, additional genes which are enriched in cell cycle regulation, FOXO, TP53, PI-3AKT, AMPK, TGFß, ERBB signaling pathways and in the regulation of gene expression, proliferation, cellular response to hypoxia, and negative regulation of the apoptotic process, the GO terms have central importance in ovarian cancer development. The aberrantly expressed miRNAs might be considered as potential biomarkers for the diagnosis of ovarian cancer after validation of these results in a larger cohort of ovarian cancer patients.

Expression of CD24 in plasma, exosome and ovarian tissue samples of serous ovarian cancer patients.

CD24 is a small molecular weight cell-surface protein and an independent marker for poor prognosis in the different type of cancers. We aimed to determine the expression of CD24 in plasma, exosomes and ovarian tissue samples of serous ovarian cancer patients. We collected tissue and blood samples from 21 cases of serous ovarian cancer and eight healthy controls. We used silica adsorption method for isolation of RNA. The cDNA was synthesized using quantitative real-time PCR. We used beta-globin as a housekeeping gene for the normalization of the data. Protein-protein and miRNA networking were analyzed. There was a significant difference in the expression of CD24 in ovarian tissue between controls and patients (0.16 ± 0.32 vs. 44.97 ± 68.06; p < 0.01), while CD24 did not show expression in each plasma and exosome samples. There was a correlation in the expression of CD24 and FIGO grading between controls and patients. CD24 expression was detected in exosomes in 38.1% of patients, mainly with FIGO III, and in their plasma in 9.5% of cases. Our network analysis shows LYN, SELP, FGR, and NPM1 proteins are interacting with CD24. Our study demonstrates higher expression of CD24 in ovarian cancer patients' tissue samples, and there is an association with FIGO classification. However, CD24 showed expression only in some cell-free plasma and exosome samples.

Circulating epithelial-mesenchymal transition-associated miRNAs are promising biomarkers in ovarian cancer.

Ovarian cancer is the fifth most common cause of cancer death among women that is mostly due to the difficulty of early diagnosis. Circulating miRNAs proved to be reliable biomarkers in various cancers. We screened 9 miRNAs, which are involved in epithelial-mesenchymal transition, in the plasma samples of patients with malignant (n = 28) or non-malignant (n = 12) ovarian tumors and disease-free healthy volunteers (n = 60) by qRT-PCR. The expression levels of miR200a, miR200b, miR200c, miR141, miR429, miR203a, miR34b (p < 0.001) and miR34a (p < 0.01) were significantly higher in the malignant samples than in healthy controls. MiR203a, miR141 (p < 0.01), miR200a and miR429 (p < 0.05) levels were also higher in malignant compared to non-malignant samples. ROC-AUC was the highest in the case of miR200c: 0.861 (95%CI = 0.776-0.947). Spearman's rank correlation analysis revealed positive correlation between the plasma levels of the studied miRNAs that was the highest between miR200b and miR200c (rs = 0.774; p < 0.001). Target analysis also suggested tight interaction between these miRNAs in the regulation of cancer development. The agreement of diagnostic tests based on miRNA levels and the standard CA125 or HE4 was weak according to Cohen's kappa values. We conclude that miR200 family members, miR34b and miR203a might be promising complementary biomarkers in ovarian cancer.

Detection of cell-free, exosomal and whole blood mitochondrial DNA copy number in plasma or whole blood of patients with serous epithelial ovarian cancer.

Ovarian tumor is one of the leading causes of cancer among women. Patients are diagnosed at an advanced stage, usually. There is a need for new specific and sensitive biomarkers. Mitochondrial DNA copy number change was observed in various cancers. Our aim was to detect mitochondrial DNA copy number in whole blood (wb-mtDNA) and in plasma (cell-free and exosome encapsulated mtDNA) in patients with serous epithelial ovarian tumor. DNA was isolated from EDTA blood and plasma obtained from 24 patients and 24 healthy controls. Exosomes were isolated from cell-free plasma, and exosome encapsulated DNA (exoDNA) was extracted. Quantitative-real-time PCR was performed with Human Mitochondrial DNA (mtDNA) Monitoring Primer Set. Kruskall­Wallis and Mann­Whitney U test were used for data analysis. Wb-mtDNA copy number was significantly different among healthy controls and patients in multiple comparison (p = 0.0090 considering FIGO stage independently, and p = 0.0048 considering early- and late-stage cancers). There was a significant decrease among early-stage, all advanced stage and all cancer patients (FIGO I: 32.5 ± 8.3, p = 0.0061; FIGO III + IV: 37.2 ± 13.7 p = 0.0139; FIGO I + III + IV: 35.6 ± 12.2, p = 0.0017) or FIGO III patients alone (32.8 ± 5.6, p = 0.00089) compared to healthy controls. We found significant increase in copy number in exosomal mtDNA in cancer patients (236.0 ± 499.0, p = 0.0155), advanced-stage cancer patients (333.0 ± 575.0, p = 0.0095), of FIGO III (362.0 ± 609.2, p = 0.0494), and FIGO IV (304.0 ± 585.0, p = 0.0393) patients alone but not in samples of FIGO I patients (10.0 ± 3.5, p = 0.3907). In multiple comparison the increase was significant considering early- and late-stage cancers (p = 0.0253). Cell-free mtDNA copy numbers were not increased significantly. We found the highest copy number of mtDNA in exosomes, followed by plasma and peripheral blood in late-stage cancer patients. We observed significant difference in wb-mtDNA copy number between healthy controls and both early- and late-stage cancer patients.

Mutation in afsR Leads to A-Factor Deficiency in Streptomyces griseus B2682.

BACKGROUND/AIMS: A-factor, a γ-butyrolactone autoregulator, in Streptomyces griseus is involved in the regulation of differentiation and antibiotic production. Here we studied the S. griseus B2682-AFN (A-factor negative) bald mutant that harbors a nonsense mutation in the afsR gene encoding a pleiotropic regulator. Our aim was to prove that this mutation is the cause of the A-factor deficiency in AFN. We also studied whether AfsR regulates A-factor production by AfsA, which is supposed to be the only specific key enzyme in A-factor biosynthesis. METHODS: Wild afsR was cloned to the pHJL401 shuttle vector and was transformed to the S. griseus AFN and B2682 strains. During phenotypic characterization, sporulation, antibiotic, protease, A-factor, and AfsA protein production were studied. RESULTS: Transformation of AFN by a wild afsR restored its phenotype including sporulation, antibiotic, extracellular protease, and A-factor production. Introduction of afsR to the B2682 wild-type strain resulted in antibiotic and extracellular protease overproduction that was accompanied with an elevated A-factor level. AfsA was detected both in AFN and B2682. CONCLUSIONS: AfsR has an effect on the regulation of A-factor production in S. griseus. The presence of AfsA is not sufficient for normal A-factor production. AfsR regulates A-factor biosynthesis independently of AfsA.

[Treatment outcome of patients with essential thrombocythemia at our department in Hungary]. / Essentialis thrombocythaemiás betegek kezelési eredményei osztályunkon.

INTRODUCTION: Essential thrombocythemia is a Philadelphia chromosome-negative chronic myeloproliferative neoplasia with a risk of bleeding and thromboembolic complications during the course of illness. Cytoreductive drugs, such as non-selective hydroxyurea or interferon as first-line and specific, megakaryocyte-thrombocyte reductive anagrelide chosen as second-line treatment in cases of adverse, intolerable effects of hydroxyurea can lower the incidence of bleeding/thrombotic episodes in patients with essential thrombocythemia. AIM: In this observational survey the effect of anagrelide was investigated in patients with essential thrombocythemia, who were first treated with hydroxyurea but failed to have clinicopathologic reponse (resistant) or were intolerant (adverse effects). METHOD: Between 2000 and 2014, 104 patients were diagnosed with essential thrombocythemia and treated first-line with hydroxyurea (weekly median dose of 7500 mg) in the haematologic outpatient department of the authors. Because of intolerance and/or resistance, hydroxyurea was changed to anagrelide (7.5 mg weekly median dose), the doses of hydroxyurea and anagrelide were adjusted to achieve clinicopathological response according to the updated criteria of the European LeukemiaNET. Effect of anagrelide as monotherapy (first- or second-line after hydroxyurea) or in combination with hydroxyurea was followed. Statistical analysis was performed using the Windows Statistical Package Program. RESULTS: Of the 104 patients with essential thrombocythemia (according to the updated WHO-ET classifications 58 patients JAK2V617F mutation positive, 46 patients negative, 15 patients calreticulin mutation negative, 6 patients MPL-1 mutation negative) 87 patients received hydroxyurea in first line, 4 patients interferon, and 13 patients acetylsalycilic acid only. Seven patients who proved to be intolerant and 22 patients who were resistant to hydroxyurea received anagrelide in second line (in 18 patients monotherapy and in 11 patients in combination with hydroxyurea), while other 5 rather young patients in first line therapy (34/104, 32.6%). In the anagrelide first line group 5 patients (100%), in the second line anagrelide monotherapy group 16 patients (88,8%), and in the combined hydroxyurea plus anagrelide group 9 patients (82.1%) achieved complete remission. The 10-year overall survival was 82.1%. In 2 patients treated with anagrelide major bleeding and in one patient myocardial infarction occurred, other serious adverse events due to anagrelide treatment were not detected. Three elder patients died from non-hematologic diseases, but leukaemic transformation was not observed. CONCLUSIONS: First or second line anagrelide therapy, combined with hydroxyurea if necessary, was able to reduce the platelet-count and the rate of complications, and to control the course of essential thrombocythemia with tolerable adverse effects.

[Treatment outcome in primary testicular non-Hodgkin lymphoma]. / Primer herelymphomás betegek kezelésével szerzett tapasztalatok.

INTRODUCTION: Primary testicular lymphoma constitutes a rare subgroup among extranodal non-Hodgkin's lymphomas. Because of its aggressive clinical behaviour due to high grade histological features developing mainly in older population, patients with this disease usually have a poor prognosis. Orchidectomy followed by combination immunochemotherapy is a traditional treatment method with a rather inferior outcome. AIM: In this retrospective survey the authors analysed the clinical presentation, pathological features and treatment results of patients with primary testicular lymphoma diagnosed and treated in their haematology centre between 2000-2012 METHOD: During this period 334 patients with aggressive non-Hodgkin's lymphomas were treated, of whom 8 patients (2.39%; age between 23 and 86 years; median, 60 years) underwent semicastration for primary testicular lymphoma (7 patients had diffuse, large B-cell lymphoma and one patient had Burkitt-like lymphoma). According to the Ann Arbor staging system a limited stage I-IIE was diagnosed in 7 patients and advanced stage was found in one patient. All but one patients were treated with rituximab added to CHOP regimen (6 or 8 cycles in every 21 or 28 days), whereas one patient received radiotherapy only. Central nervous system intrathecal prophylaxis was used in one case and no preventive irradiation of the contralateral testis was used. RESULTS: With a median follow-up of 50 months complete remission was observed in 7 patients. However, two patients died (one due to progression and one in remission from pulmonary solid tumour). Complete remission rate proved to be 87.5%, disease-free survival was between 13 and 152 months (median 38 months) and overall survival rates were between 17 and 156 months (median 43 months). The 5-year disease-free and overall survival rates were 37.5 %. CONCLUSIONS: The relatively favourable treatment outcome could be mainly explained by the high number of patients with early-stage of the disease, early surgical removal of testicular lymphomas and the use if immunochemotherapy. This therapeutic regimen was effective to prevent localized and distant relapses. Despite omission of regular prophylaxis of the central nervous system, no relapse was detected.

[Treatment outcome of immune thrombocytopenia]. / Immun thrombocytopeniás betegek kezelésével szerzett tapasztalataink.

INTRODUCTION: Treatment of immune thrombocytopenia is sometimes difficult and needs personal setting. According to evidence-based guidelines, corticosteroids are suggested for first-line treatment. In case of corticosteroid ineffectiveness, second-line therapeutic options (splenectomy, immunosuppressive drugs and, recently, thrombopoietin-mimetics) may result in beneficial therapeutic effect. AIMS: The aim of the authors was to examine the clinicopathological data, disease course, treatment results, and the effectiveness of novel drugs in patients with immune thrombocytopenia. PATIENTS AND METHODS: The authors retrospectively analysed the files of 79 immune thrombocytopenic patients (26 males and 53 females) diagnosed and treated at the hematologic in- and outpatient units of the Markusovszky Hospital, County Vas, Hungary between January 1, 2000 and December 31, 2011. Remission rates, disease-free and overall survivals in response to corticosteroids (first-line treatment), after splenectomy (in cases when corticosteroids proved to be ineffective) and following second-line treatment were analysed. Survival curves were constructed using statistical software programs. RESULTS: Of the 79 patients during a median follow-up of 66 months (min. 3, max. 144 months), 28 patients receiving first-line corticosteroids achieved complete remission and remained in a prolonged disease-free condition (35.4%; median disease-free survival 75.5 months; min. 2, max. 140 months). Thirty-eight patients underwent splenectomy after ineffective treatment with corticosteroids or other immunosuppressive (48.0%; median disease-free survival 94.2 months; min. 6, max. 136 months). Surgical complications occurred in 2 cases, while postoperative and late infections were absent. Five patients died but death was not related to immune thrombocytemia. Second-line treatment was applied in 13 patients (16.4%) and among these patients relapse of immune thrombocytopenia after splenectomy was observed in 6 patients. Favourable effects of both conventional (immunosuppressive) and novel treatments (rituximab, thrombopoietin-mimetics) were also detected. CONCLUSIONS: More than two-thirds of patients with immune thrombocytopenia responded to corticosteroids or to splenectomy and achieved prolonged disease-free remission. Novel drugs (rituximab, thrombopoietin-mimetics) applied only in few cases produced also favourable results in patients not responding to corticosteroids and splenectomy.

[Significance of the JAK2V617F mutation in patients with chronic myeloproliferative neoplasia]. / A JAK2V617F-mutáció jelentosége krónikus myeloproliferativ neoplasiás betegeinkben.

UNLABELLED: In Philadelphia chromosome-negative chronic myeloproliferative neoplasia, i.e. polycythemia vera, essential thrombocythemia and primary idiopathic myelofibrosis enhanced risk of thrombosis could be connected with Janus kinase 2 gene mutation occurring in various frequency in these diseases (JAK2V617F). Since 2002 the presence of JAK2 mutation in chronic myeloproliferative neoplasia has been regularly detected. AIMS: In a retrospective survey the possible connection between JAK2 mutation and thrombosis was analyzed in patients with chronic myeloproliferative neoplasia subgroups cared and treated in their hospital and outpatient departments. PATIENTS AND METHODS: Between 2007-2010 peripheral blood samples of 171 patients with chronic myeloproliferative neoplasia (68 patients of polycythemia vera, 84 of essential thrombocythemia and 19 ones with primary idiopathic myelofibrosis) were sent to several molecular biological laboratories, where V617F mutation from DNA specimens was detected by allele-specific polymerase chain reaction, as well. Thromboembolic complications (arterial, i.e. cerebro-and cardiovascular and venous thrombosis) occurred during course of illness of patients were registered. Statistical analysis was made by statistical software program for Windows. RESULTS: JAK2 mutation in 53 patients with polycythemia vera (77.9%) was detected, whilst in essential thrombocythemia 55 patients (65.4%) and in primary idiopathic myelofibrosis 7 patients (36.8%) proved to be JAK2 positive. In 18 JAK2 positive patients of polycythemia vera thromboembolic episodes were observed (18/53, 33.9%), whilst in essential thrombocythemia JAK2 mutational status was accompanied with thromboembolic events in 17/55 patients (30.9%). In the 7 JAK2 positive ones with primary idiopathic myelofibrosis thrombotic complication did not occurred. However, in JAK2 negative cases thrombotic events could also be detected (from 10 JAK2 negative patients with polycythemia vera in four ones, and in six with JAK2 negative 23 essential thrombocythemic patients. CONCLUSIONS: Incidence of the JAK2 mutation in their patients with chronic myeloproliferative neoplasia subgroups mainly corresponds to the literary data. Thrombosis ensued both in JAK positive polycythemia vera and essential thrombocythemia cases occurred nearly in the same number, but the incidence of thrombosis ensued in JAK2 negative cases did not differ significantly from the JAK2 positive patients. From these results it could be suggested that the presence or absence of JAK2 mutation in the development of thrombosis has no predictive value in patients with chronic myeloproliferative neoplasia.

[Therapeutic management of central nervous system lymphomas in a single hematological institute]. / Központi idegrendszeri lymphomás betegek kezelése osztályunkon.

UNLABELLED: Primary central nervous system lymphoma is defined as an extranodal lymphoma arising in the central nervous system in the absence of systemic disease. Because of their rare occurrence among lymphomas, optimal treatment could hardly be established. AIMS: In this retrospective survey we analyzed the result of combined treatment (systemic and intrathecal chemotherapy followed by consolidation radiotherapy) in patients with primary or relapsed central nervous system lymphomas diagnosed and treated in our hematological department between 1998-2009. PATIENTS AND METHODS: During this period (mean follow-up of 13.2 months) from 427 patients with newly diagnosed non-Hodgkin's lymphomas, 22 primary central nervous system lymphoma was diagnosed (5.15%, 16 cerebral and 6 spinal cord lymphoma cases). Significant male predominance (14:8) was registered with an age distribution of 34-77 years (mean = 60.7, median = 64 years). No patients were immunocompromised. All central nervous system lymphoma specimens taken with neurosurgical resection or stereotaxic biopsies were confirmed histopathologically. All cerebral lymphoma cases proved to be diffuse large B-cell of origin, while in epidural lymphomas low grade subtypes also occurred. Epidural lymphomas were treated with local radiotherapy (30-40 Gy), except for patients with follicular lymphomas getting rituximab-containing polychemotherapy (R + CHOP regimen) before irradiation. In cerebral lymphoma (every patients had supratentorial localization) the following combined therapy protocol was used: up to three courses of high dose methotrexate (HD MTX 3g/m 2 in a single dose for 4 hours lasting drop-infusion) were given at 4-week intervals, followed by leucovorin-rescue 24 hours after MTX infusion. Intrathecal combination of methotrexate, cytosin-arabinosid and dexamethasone was given three times after HD MTX infusion. In complete response after chemotherapy (evaluated by cranial MRI or CT, PET/CT), whole-brain irradiation was used in a total dose of 30 Gy. In case of partial response, boost irradiation for the tumor bed was also given. In relapse or resistant cases, salvage regimen was applied: HD MTX course combined with high dose cytosin-arabinosid (HD Ara-C) 3g/m 2 /dose b.i.d. over 4 h c.i., repeated in three cycles every four weeks. RESULTS: Complete remission has been achieved in 9 patients with cerebral and in 4 patients with spinal cord lymphoma (13/22; 59.0%), however, one relapsed patient became resistant and later expired, despite salvage therapy. Primarily 9 patients were not evaluable for response: 5 received only one or two HD MTX because of side effects, 4 patients died due to progression of the disease. Mean of the overall survival (OS) in cerebral lymphoma was 19.5 (3-46, median of 10) months, in epidural group 14.1 (2-76, median of 5) months, whilst mean time to progression (TTP) was 4.5 (2-6.5, median of 4 months). The 2-year survival for all patients was 50%. Acute toxicity of chemotherapy was usually hematological, moreover, in 8 patients impaired renal function and sepsis developed. No serious adverse effect of radiotherapy could be detected. CONCLUSION: In primary central nervous system lymphoma, basic treatment HD methotrexate together with intrathecal combination of methotrexate + cytosin-arabinosid + dexamethasone followed by whole-brain irradiation of at least 30 Gy could produce a medium response rate in our study. In case of relapse or progression, other salvage regimens containing HD Ara-C alternating with HD MTX could reduce the treatment failure, as well. After therapy PET/CT was negative in five patients with prolonged disease-free survival.

[Complex analysis of prognostic factors in chronic lymphocytic leukemia]. / Prognosztikai faktorok komplex vizsgálata krónikus lymphocytás leukémiában.

INTRODUCTION: Many new prognostic factors established in recent years in chronic lymphocytic leukemia. May help predicting survival. AIMS: The goal of the present study was to determine the frequency and the correlation of these novel prognostic factors in samples of 419 leukemia patients. METHODS: The mutation status of the IgH gene was evaluated in 160 cases. RESULTS: In 62% of cases, non-mutated IgH gene was found, the heavy chain family usage was different in mutated and non-mutated cases. The CD38 expression demonstrated 78% concordance with the mutation status, the ZAP-70 expression failed to show any correlation. Cytogenetic abnormalities were seen in 76% of cases, the most frequent were del(13q) (57%), trisomy 12 (15%), del(11q) (12%) and del(17p) (6%). 95% of cases with del(11q) harbored non-mutated, 74% of cases with del(13q) as the sole anomaly demonstrated mutated IgH genes. CONCLUSIONS: The parameters analysed are not independent of each other, utilization of them in the clinical routine needs careful planning.

Absence of microdeletions in the azoospermia-factor region of the Y-chromosome in Viennese men seeking assisted reproduction.

PURPOSE: The azoospermia-factor region of the Y-chromosome is essential for spermatogenesis in humans. In the literature, a wide range is given for the frequency of microdeletions in this region. The purpose of this study was to evaluate our own population of patients. METHODS: During a two-year period at Vienna Medical School, all male patients (n = 383) seeking assisted reproduction were screened for microdeletions. Thirty-three men had azoospermia and 154 severe oligozoospermia. Genomic DNA was prepared from peripheral lymphocytes and polymerase chain reaction analysis of the azoospermia-factor region was performed using the Promega kit. RESULTS: No case tested positive for azoospermia-factor microdeletions. In all cases amplification of 18 non-polymorphic sequence tagged sites was obtained. CONCLUSIONS: Y-chromosome microdeletions do not seem to be an important factor for male infertility in our patients. This suggests that screening should be restricted to men with azoospermia or severe oligozoospermia only.