A multicentre, open-label, phase-I/randomised phase-II study to evaluate safety, pharmacokinetics, and efficacy of nintedanib vs. sorafenib in European patients with advanced hepatocellular carcinoma.

BACKGROUND: This multicentre, open-label, phase-I/randomised phase-II trial evaluated safety, pharmacokinetics, maximum-tolerated-dose (MTD) per dose-limiting toxicities (DLTs), and efficacy of nintedanib vs. sorafenib in European patients with unresectable advanced hepatocellular carcinoma (aHCC). METHODS: Phase I: Patients were stratified into two groups per baseline aminotransferase/alanine aminotransferase and Child-Pugh score; MTD was determined. Phase II: Patients were randomised 2:1 to nintedanib (MTD) or sorafenib (400-mg bid) in 28-day cycles until intolerance or disease progression. Time-to-progression (TTP, primary endpoint), overall survival (OS) and progression-free survival (PFS) were determined. RESULTS: Phase-I: no DLTs observed; nintedanib MTD in both groups was 200 mg bid. Phase-II: patients (N = 93) were randomised to nintedanib (n = 62) or sorafenib (n = 31); TTP was 5.5 vs. 4.6 months (HR = 1.44 [95% CI, 0.81-2.57]), OS was 11.9 vs. 11.4 months (HR = 0.88 [95% CI, 0.52-1.47]), PFS was 5.3 vs. 3.9 months (HR = 1.35 [95% CI, 0.78-2.34]), respectively (all medians). Dose intensity and tolerability favoured nintedanib. Fewer patients on nintedanib (87.1%) vs. sorafenib (96.8%) had drug-related adverse events (AEs) or grade ≥ 3 AEs (67.7% vs. 90.3%), but more patients on nintedanib (28 [45.2%]) had AEs leading to drug discontinuation than did those on sorafenib (7 [22.6%]). CONCLUSIONS: Nintedanib may have similar efficacy to sorafenib in aHCC.

A randomized, double-blind, placebo-controlled phase II study to assess the efficacy and safety of mapatumumab with sorafenib in patients with advanced hepatocellular carcinoma.

BACKGROUND: This randomized, double-blind, placebo-controlled, phase II study evaluated the efficacy and safety of mapatumumab (a human agonistic monoclonal antibody against tumor necrosis factor-related apoptosis-inducing ligand receptor 1) in combination with sorafenib in patients with advanced hepatocellular carcinoma (HCC). PATIENTS AND METHODS: Patients with advanced HCC (stratified by Barcelona Clinic Liver Cancer stage and Eastern Cooperative Oncology Group performance status) were randomized 1:1 to receive sorafenib (400 mg, twice daily per 21-day cycle) and either placebo (placebo-sorafenib arm) or mapatumumab (30 mg/kg on day 1 per 21-day cycle; mapatumumab-sorafenib arm). The primary end point was time to (radiologic) progression (TTP), assessed by blinded independent central review. Key secondary end points included progression-free survival, overall survival, and objective response. RESULTS: In total, 101 patients were randomized (placebo-sorafenib arm: N = 51; mapatumumab-sorafenib arm: N = 50). There was no significant difference in median TTP between both arms [5.6 versus 4.1 months, respectively; adjusted hazard ratio (one-sided 90% confidence interval) 1.192 (0-1.737)]. No mapatumumab-related benefit was identified when TTP was evaluated in the stratified subgroups. The addition of mapatumumab to sorafenib did not demonstrate improvement in the secondary efficacy end points. The reported frequency of adverse events (AEs) and serious AEs was comparable in both treatment arms. CONCLUSIONS: The addition of mapatumumab to sorafenib did not improve TTP or other efficacy end points, nor did it substantially change the toxicity profile of sorafenib in patients with advanced HCC. Based on these results, further development of the combination of mapatumumab and sorafenib in HCC is not planned.

Efficacy and safety of a nanofiltered liquid intravenous immunoglobulin product in patients with primary immunodeficiency and idiopathic thrombocytopenic purpura.

BACKGROUND AND OBJECTIVES: In the production process of a new 5% liquid intravenous immunoglobulin (IVIG-L) product (Nanogam(®) ), a combined pepsin/pH 4·4 treatment/15-nm filtration (pH 4·4/15NF) step and a solvent-detergent (SD) treatment step were incorporated to improve the virus inactivating/reducing capacity of the manufacturing process. Two prospective uncontrolled multicentre studies were performed to evaluate the safety and efficacy of this product. MATERIALS AND METHODS: Efficacy, including pharmacokinetics, of IVIG-L was studied for 6 months in 18 primary immunodeficiency (PID) patients, succeeded by a long-term follow-up study (mean 2·2 years, n=17). Second, in 24 patients with idiopathic thrombocytopenic purpura (ITP), IVIG-L was studied for efficacy for 14 days. In both studies, adverse events and vital signs were recorded to study safety. RESULTS: In PID patients treated with IVIG-L, 0·60 and 0·38 severe infections per patient per year were reported during, respectively, the short-term and long-term follow-up. Pharmacokinetic studies resulted in an IgG half-life of 30·9 ± 11·3 days and a mean IgG trough level of 6·8 ± 1·2 g/l. In the ITP study, all patients showed an increase in platelet counts after infusion with IVIG-L, and 20/24 patients responded with a platelet count >50 × 10(9) /l (83·3%) within 1 week. IVIG-L infusions did not cause clinical relevant changes in laboratory parameters or vital signs. CONCLUSIONS: In clinical studies, IVIG-L (Nanogam®) demonstrated to be efficacious, well tolerated and safe.

Clinicopathological characteristics of metaplastic breast cancer - analysis of the basic immunohistochemical profile and comparison with other invasive breast cancer types.

INTRODUCTION: Metaplastic breast cancer (MpBC) is a rare but aggressive type of breast cancer accounting for 0.25-1% of all diagnosed invasive breast cancers. Morphologically, it is characterized by differentiation of the neoplastic epithelium into squamous cells and/or mesenchymal-looking tissue. MATERIAL AND METHODS: We analyzed 13 MpBCs selected from the group of 1122 invasive breast cancers. Histopathological examination and analysis of estrogen (ER), progesterone (PR) and HER2 receptors expression in MpBC patients and their comparison to other types of invasive breast cancer has been performed. RESULTS: 13 MpBC cases represented 1.16% of the 1122 invasive breast cancers. The MpBC group presented with a significantly larger tumor size (≥T2, 69% versus 49%, p < 0.001) and with higher grade of histological malignancy (G1-G3) (p < 0.001). MpBC group had significantly more cases with no hormone receptors (ER, PR) and HER2 overexpression/gene amplification compared with the other invasive breast cancer types group (ER-, 69% versus 23%, p < 0.001; PR-, 69% versus 28%, p < 0.001; HER2 0/1+, 93% versus 82%, p = 0.019). Most MpBCs (62%) were triple-negative. We found a correlation between hormone receptors expression and lymph node metastasis (p < 0.001). The analysis of the HER2 expression allowed us to find correlation between its expression and tumor histological grade (G1-G3) (p < 0.001), tumor size (T1a-T4) (p < 0.001) and lymph node metastasis (pN0-pN4) (p < 0.001) in MpBCs. DISCUSSION: MpBCs are usually larger at primary diagnosis and most of MpBCs present with other poor prognostic indicators and show lack of steroid hormone receptors expression as well as HER2. Hormone receptor status and HER2 expression seems to correlate with histological grade of malignancy (G1-G3), tumor size (T1a-T4) and regional lymph node involvement (pN0-pN4) and these features are directly related to MpBC malignancy.

Effects of the flavonoid baicalin and its metabolite baicalein on androgen receptor expression, cell cycle progression and apoptosis of prostate cancer cell lines.

Recent studies on the Chinese herbal medicine PC SPES showed biological activities against prostate cancer in vitro, in vivo and in patients with advanced stages of the disease. In investigating its mode of action, we have isolated a few of the active compounds. Among them, baicalin was the most abundant (about 6%) in the ethanol extract of PC SPES, as determined by HPLC. Baicalin is known to be converted in vivo to baicalein by the cleavage of the glycoside moiety. Therefore, it is useful to compare their activities in vitro. The effects of baicalin and baicalein were studied in androgen-positive and -negative human prostate cancer lines LNCaP and JCA-1, respectively. Inhibition of cell growth by 50% (ED(50)) in LNCaP cells was seen at concentrations of 60.8 +/- 3.2 and 29.8 +/- 2.2 microM baicalin and baicalein, respectively. More potent growth inhibitory effects were observed in androgen-negative JCA-1 cells, for which the ED(50) values for baicalin and baicalein were 46.8 +/- 0.7 and 17.7 +/- 3.4, respectively. Thus, it appears that cell growth inhibition by these flavonoids is independent of androgen receptor status. Both agents (1) caused an apparent accumulation of cells in G(1) at the ED(50) concentration, (2) induced apoptosis at higher concentrations, and (3) decreased expression of the androgen receptor in LNCaP cells.

Enhanced in vitro cytotoxicity and cytostasis of the combination of onconase with a proteasome inhibitor.

In proliferating cells the turnover rate of proteins responsible for regulation of the cell cycle progression, namely cyclins and inhibitors of the cyclin-dependent kinases (CDKs) and phosphatases, is rapid and their cellular level is modulated at the transcriptional, translational and/or degradation (via proteasome pathway) stages. Inhibition of proteasome function results in accumulation of rapidly turning over proteins and, thus, causes an imbalance of the cell cycle regulatory components, and loss of their regulatory function. Indeed, it has been shown that proteasome inhibitors perturb the cell cycle progression. Onconase, a novel RNase which has anti-tumor activity and is in clinical trials, has previously been shown to suppress protein synthesis, presumably by degradation of intracellular RNA, preferentially tRNA. By interfering with regulation of expression of cyclins and/or CDK-inhibitors, onconase also may induce the imbalance of these proteins and potentiate the effect of proteasome inhibitors. In the present study, we observed that the combinations of onconase with peptide-aldehyde inhibitors of calpain and proteasome such as the N-acetyl-leucinyl-leucinyl-norleucinal (LLnL) and the N-acetyl-leucinyl-valinyl-phenylalaninal (LVP), but not N-acetyl-leucinyl-leucinyl-methioninal (LLM), were synergistic in suppressing cell proliferation and inducing apoptosis in three human tumor cell lines: A-549 lung adenocarcinoma, DU-145 prostatic carcinoma, and MDA-MB-231 breast carcinoma. The observed cytotoxicity may also be a result of prevention of the induction of the 'survival' genes by the nuclear factor kappaB (NFkappaB) by onconase and proteasome inhibitors. The data indicate that such combinations should be further tested as potential anti-cancer regimens.

Potentiation of tumor necrosis factor induced apoptosis by onconase.

Onconase (ONC) a ribonuclease from amphibian oocytes is cytostatic and cytotoxic to many human tumor lines, shows in vivo antitumor activity in mouse tumor models and is in Phase III clinical trials. The mechanism of antitumor activity of ONC is presumed to be due to its internalization, degradation of intracellular RNA and suppression of protein synthesis. Since apoptosis triggered by TNF-alpha is known to be potentiated by inhibitors of protein synthesis, we have hypothesized that it also may be potentiated by ONC. Indeed, preincubation of U-937 or HL-60 leukemic cells with 0.17 microM ONC rendered them more sensitive to induction of apoptosis by TNF-alpha or antibody to CD95 (Fas). The mechanism by which ONC amplifies the effect of TNF-alpha may involve suppression of induction of the survival genes whose expression is triggered by activation of NFkB by this factor.

Differences in induction of p53, p21WAF1 and apoptosis in relation to cell cycle phase of MCF-7 cells treated with camptothecin.

The DNA topoisomerase I (topI) inhibitor camptothecin (CPT), stabilizes so-called cleavable complexes which consist of topI covalently attached to 3' OH ends of DNA nicks. Collisions between the progressing DNA replication forks (occurring in S phase cells) or between the transcription driven RNA polymerase molecules (occurring in G1, S and G2 cells) and these complexes convert the latter into secondary DNA lesions which are unrepairable and lethal to the cell. Changes induced by CPT in the level of the tumor suppressor p53, cyclin-dependent kinase inhibitor p21WAF1 and proapoptotic protein Bax (all detected immunocytochemically), were measured separately in the nucleus and cytoplasm of individual human breast carcinoma MCF-7 cells by laser scanning cytometry (LSC) in relation to cell cycle position and induction of apoptosis. The initial transient cell arrest at the G1 checkpoint seen at 8-16 h of treatment with 0.15 microM CPT was accompanied by the rapid accumulation of p53 (preventable by cycloheximide) in the nucleus; the rise (>20-fold) in p53 was maximal for S phase cells. The magnitude of the nuclear p53 increase induced by CPT, at maximum, was 2-fold higher than that induced by the proteasome inhibitor N-acetyl-Leu-Leu-norleucinal (LLnL). While the accumulation of p53 was seen in all phases of the cycle, only G1 cells responded by induction ( approximately 60-fold increase) of p21WAF1. Inhibition of DNA replication by aphidicolin prevented the accumulation of p53 in S and G2/M but had no effect on its induction in G1 cells. Perturbation of cell progression through S phase was seen between 24-72 h of treatment, and it coincided with induction of Bax and apoptosis (both maximal in S phase cells). Thus, the changes observed in S phase cells (nuclear accumulation of p53 preventable by aphidicolin, induction of Bax, apoptosis), triggered by the collisions of DNA replication forks with the CPT-induced lesions, were distinct from the changes in G1 (nuclear p53 accumulation unaffected by aphidicolin, induction of p21WAF1) presumably triggered by collisions of RNA polymerase with the CPT-lesions. Great heterogeneity in expression of p53 and p21WAF1 of the G1 cell population in response to CPT was observed, which may reflect the intercellular variability in the rate of transcription (i.e., frequencies of collisions of RNA polymerase with the lesions). Thus, differences in the transcriptional activity of G1 cells may play a role in their sensitivity to CPT and similar topI inhibitors.

Unique analytical capabilities of laser scanning cytometry (LSC) that complement flow cytometry.

Flow cytometry has become an indispensable instrumentation in many disciplines of biology and medicine. There are some limitations of flow cytometry, inherent to the fact that the cells are measured in flow, which limit its usefulness in some applications. The microscope-based laser scanning cytometer (LSC) has many features similar to flow cytometry but few restrictions of the latter and therefore it is useful in many new applications. This review briefly outlines the applications that are unique to LSC, particularly related to its morphometric capabilities and the possibility of cell relocation. Potential future applications of LSC are also discussed.

[Skin changes in Hodgkin's disease. Case report]. / Zmiany skórne w przebiegu ziarnicy zlosliwej. Opis przypadku.

A case of Hodgkin's disease was observed in the Warsaw Institute of Haematology. Involvement of the skin developed ++during uncontrolled and refractory pathological process, despite multiple changes of cytostatic programmes. The histological type of the lesions, nodular sclerosis, suggested a milder course of the disease.

[Detection of minimal residual disease in acute leukemias by immunophenotyping of bone marrow cells]. / Wykrywanie minimalnej choroby resztkowej w ostrych bialaczkach metoda immunofenotypowania komórek szpiku kostnego.

Minimal residual disease (MRD) can be detected only by sophisticated methods, e.g. immunologic. The purpose of the study was to detect MRD in leukemia patients (AL) being in hematological and clinical remission (CR) by immunophenotyping of bone marrow cells and to define the role of the immunologic method in the detection of MRD. Bone marrow samples from 31 patients with ALL and AML and from 18 volunteers were taken several times. Staining of the cells was performed by double color immunofluorescence (IF) and double color immunoenzyme assay (APAAP). The detection of MRD was done by the comparison of the percent of positive cells between AL and normal cases. Each pair of the antigens (CD) was estimated in its usefulness in MRD detection. The statistical analyses were evaluated with a t-Student's test.

[Clinical significance and prognostic detection of minimal residual disease in acute leukemias]. / Znaczenie kliniczne i prognostyczne wykrycia minimalnej choroby resztkowej w ostrych bialaczkach.

The term minimal residual disease (MRD) describes leukemia cells present in bone marrow at a level below that detectable by conventional methodology in patients being in complete hematological and clinical remission (CR). The purpose of the study was, the detection of MRD in leukemia patients (AL-MRD+), the correlation between AL-MRD+ and the chosen hematological and clinical parameters and the follow up, and the prognosis. Bone marrow samples from 31 patients with ALL and AML, and from 18 volunteers were taken several times. Staining of cells was performed by double color immunofluorescence (IF), using flow cytometer and double color immunoenzyme assay (APAAP). The statistical analysis were evaluated with a t-Student's test and chi-squared test.

[Effectiveness of interferon alfa in different stages of thrombocythemia (preliminary report)]. / Skutecznosc interferonu-alfa w róznych stanach nadplytkowosci (Doniesienie wstepne).

In 6 women aged 38 to 68 years with thrombocythaemia during chronic myeloid leukaemia (4 cases), myelofibrosis (1 case), and idiopathic thrombocythaemia (1 case) the effects of recombinant human alpha-interferon (Intron A, rh IFN alpha -2b, Schering) were studied. The drug was given to all patients subcutaneously in one daily dose of 3 x 10(6) u, every day for 3 weeks, and then in the same doses twice weekly for 2 weeks (5 cases) and for 14 weeks (1 case). Intron A caused in all cases a fall of peripheral blood platelet count by 37% to 65.5% (mean 50%) in relation to the initial count (532 - 1,453 x 10(9)/l). The fall of the platelet count occurred usually after 7-10 days of this treatment, and the lowest count was noted usually after 24 days (10 to 42 days). During the treatment in 4 cases the peripheral leucocyte count dropped as well by 20-70%. In no cases exacerbation of chronic myeloid leukaemia was noted, and in the patient with myelofibrosis the enlarged spleen shrunk somewhat. These results of treatment and follow-up of patients with thrombocythaemia treated with Intron A indicate a significant although short-lasting effect of platelet count fall limited, however, to the time of the treatment. Side effects of the drug included mainly febrile conditions, myalgia and arthralgia.

[Clinical and hematological analysis of acute granulocytopenia]. / Analiza kliniczna i hematologiczna ostrych granulocytopenii.

Retrospective analysis of 13 patients treated for the acute granulocytopenia at the Institute of Haematology for the last 10 years was carried out. Drugs were the most common causative factor. Comparing the results of the similar analysis several years ago in which the significant role was played by chloramphenicol, non-steroidal anti-inflammatory agents were the main cause. Infective complications were seen in all analysed patients. Broad spectrum antibiotics were used in all patients. Glycocorticosteroids were given to 9 patients while leucocytic concentrates to 7 patients. Remission was achieved in 12 patients, one female patient with the history of exposition to plant protection agents and increased levels of chlorinated aromatic hydrocarbons--died. Intensive antibacterial therapy significantly lowered mortality of the analysed patients in comparison with the similar group analysed earlier. Recurrence of granulocytopenia was seen in 5 patients following the contact with causative agent. Recurrencies were finished with remission.

[Chloroma--a rare symptom of acute leukemia]. / Zieleniak (chloroma)--rzadki objawy ostrej bialaczki.

Chloroma is a tumour composed of immature granulocytes localized in extramedullary tissues. Chloroma is noted in the course of the acute non-lymphoblastic leukemia (ANLL), chronic myeloid leukemia (CML), and in myelodysplastic syndromes (MDS) during their transformation in the acute leukemias. Due to chloroma infrequency, two cases seen at the Department of Hematology, Medical Academy in Warsaw are presented. A survey of the actual literature is included.

Induction of apoptosis in bone marrow cells after treatment of mice with WR-2721 and gamma-rays: relationship to the cell cycle.

Apoptosis and cell proliferation are accepted to be responsible for the maintenance of homeostasis in the hematopoietic system. Understanding of the mechanisms of action of the aminothiols and ionizing radiation on normal hematopoietic cells requires determination of the correlation between apoptotic cell death and cell cycle distribution. The effects of WR-2721 ((S)-2-/3-aminopropylamino/ethylphosphorothioic acid; Amifostine) and 60Co gamma-rays on apoptosis and cell cycle progression in the mouse bone marrow were determined. Adult male Swiss mice were exposed to 6 Gy gamma-rays only, or pretreated with WR-2721, at a dose of 400 mg/kg body weight, 30 min before gamma-irradiation. The laser scanning cytometry APO-BRDU assay based on simultaneous analysis of cellular DNA content and the in situ detection of DNA strand breaks was used to identify apoptotic cells and to reveal the cell cycle position of apoptotic and nonapoptotic cells. Temporary changes in the frequency of apoptotic cells with fluorescein isothiocyanate (FITC) labeling of DNA strand breaks, and all bone marrow cells including apoptotic and nonapoptotic ones, whose DNA stained with propidium iodide, were observed in the particular phases of the cell cycle throughout the 96-h period after WR-2721 application and gamma-irradiation. The cell cycle phase specificity of WR-2721 and 60Co gamma-irradiation was shown in terms of induction of apoptosis in bone marrow cells. The patterns of alterations in the frequency of apoptotic cells and all bone marrow cells with respect to their cell cycle position were dependent on the agent(s) applied and the time interval after treatment of mice with WR-2721 and/or gamma-rays. A modulatory, suppressive action of WR-2721 on apoptosis induction and the cell cycle perturbation caused in normal cells of the mouse bone marrow by gamma-rays was found.

Activation of nuclear factor kappa B (NF-kappaB) assayed by laser scanning cytometry (LSC).

Nuclear factor kappa B (NF-kappaB)/rel is the family of ubiquitous transcriptional activators involved in regulation of diverse immune and inflammatory responses. It also plays a role in control of cell growth and apoptosis. In its inactive form NF-kappaB remains in the cytoplasm sequestered through interaction with IkappaB protein. Rapid translocation of NF-kappaB from cytoplasm to nucleus that occurs in response to extracellular signals is considered to be a hallmark feature of its activation. The translocation of NF-kappaB in HL-60, U-937 and Jurkat leukemic cells as well as in human fibroblasts induced by tumor necrosis factor alpha (TNF-alpha) or phorbol myristate acetate (PMA) was presently measured by laser scanning cytometry (LSC). NF-kappaB was detected immunocytochemically with FITC-tagged antibody and its presence in the nucleus vis-a-vis cytoplasm was monitored by measuring the green fluorescence integrated over the nucleus, which was counterstained with propidium iodide (PI), and over the cytoplasm, respectively. Activation of NF-kappaB led to a rapid increase in NF-kappaB-associated fluorescence measured over the nucleus (FN) concomitant with a decrease in fluorescence over the cytoplasm (F(C)), which was reflected by an increase in F(N)/F(C) ratio. This rapid assay of NF-kappaB activation can be combined with morphological identification of the activated cells or with their immunophenotype. Bivariate analysis of NF-kappaB expression versus cellular DNA content makes it possible to correlate its activation with the cell cycle position. The described method has a potential to be used as a functional assay to monitor intracellular translocation of other transcriptional activators such as p53 tumor suppressor protein or signal transduction molecules.

[Nocturnal paroxysmal hemoglobinuria--case report]. / Nocna napadowa hemoglobinuria--opis dwóch przypadków.

Paroxysmal nocturnal hemoglobinuria (PNH)-a case report. We present clinical and postmortem findings in patient with PNH who developed cerebral thrombosis. The pathogenesis of the PNH is discussed.