Undifferentiated versus retinoic acid-differentiated SH-SY5Y cells in investigation of markers of neural function in toxicological research.

The SH-SY5Y human neuroblastoma cell line is a standard in vitro experimental model of neuronal-like cells used in neuroscience and toxicological research. These cells can be differentiated into mature neurons, most commonly using retinoic acid (RA). Despite differences in characteristics, both undifferentiated and differentiated SH-SY5Y cells are used in research. However, due to uncertainties regarding the expression of specific markers of neural function in each culture, there is no definite conclusion on which culture is better suited for (neuro)toxicological and/or neuroscience investigations. To address this dilemma, we investigated the basal expression/activity of the key elements of acetylcholine, dopamine, serotonin, and GABA neurotransmitter pathways, along with the elements involved in exocytosis of neurotransmitters, and neuron electrophysiological activity in undifferentiated and in RA-differentiated SH-SY5Y cells using a six-day differentiation protocol. Our findings revealed that both SH-SY5Y cell types are functionally active. While undifferentiated SH-SY5Y cells exhibited greater multipotency in the expression of tested markers, most of those markers expressed in both cell types showed higher expression levels in RA-differentiated SH-SY5Y cells. Our results suggest that the six-day differentiation protocol with RA induces maturation, but not differentiation of the cells into specific neuron phenotype. The greater multipotency of undifferentiated cells in neural markers expression, together with their higher sensitivity to xenobiotic exposure and more simple cultivation protocols, make them a better candidate for high throughput toxicological screenings. Differentiated neurons are better suited for neuroscience researches that require higher expression of more specific neural markers and the specific types of neural cells.

Assessment of caffeine neurotoxicity using novel biomarkers of neural function in SH-SY5Y cells - Is there a need for environmental concern?

Worldwide usage of caffeine results in its constant release into the aquatic environment and growing concerns related to associated risks. We assessed (neuro)toxicity of environmentally relevant concentrations of caffeine, using novel biomarkers of neural function in SH-SY5Y cells and markers of general toxicity also in HepG2 cells. The RQ-PCR analyses showed that caffeine disturbs the expression of genes encoding several key elements of neurotransmitter pathways, with the most prominent responses observed for serotonin receptor 3A, dopamine receptor D2, monoamine oxidase B and GABA-transaminase. Expression of genes encoding synaptotagmin 10 involved in exocytosis of neurotransmitters and ATPase Na+/K+ transporting subunit alpha 3 was also disturbed. Caffeine stimulated the activity of monoamine oxidase, while cytotoxicity and effects on mitochondrial membrane potential were not observed. Our study points out the new possible molecular targets of caffeine and suggests that the raising concerns related to its growing environmental presence are justified.

Safety and efficacy of Elancoban® G200 (monensin sodium) for chickens for fattening, chickens reared for laying and turkeys.

The feed additive Elancoban® G200, containing the active substance monensin sodium, an ionophore anticoccidial, is intended to control coccidiosis in chickens for fattening, chickens reared for laying and turkeys. The FEEDAP Panel cannot conclude on the safety of the additive for the target species, consumer, user and environment with regard to the safety of the production strain. The following conclusions apply to monensin sodium included in the additive. Based on the available data set, the FEEDAP Panel cannot conclude on the safety of Elancoban® G200 for chickens for fattening. Monensin sodium is safe for turkeys for fattening with a margin of safety of 1.5. Monensin sodium is not genotoxic and not carcinogenic. The pharmacological no observed adverse effect level (NOAEL) of 0.345 mg monensin sodium/kg body weight (bw) per day was identified in dog. The acceptable daily intake (ADI) derived from this NOAEL is 0.003 mg monensin sodium/kg bw applying an uncertainty factor of 100. Elancoban® G200 is safe for the consumer. The existing maximum residue limits (MRLs) ensure consumer safety, provided that the withdrawal period of 1 day is respected. Elancoban® G200 is very irritant for the eye, but it is not a skin irritant. Elancoban® G200 should be regarded as a potential skin and respiratory sensitiser. Inhalation exposure is considered a risk to persons handling the additive. Elancoban® G200 does not pose a risk for the terrestrial compartment, the aquatic compartment and the sediment. The bioaccumulation potential of monensin in the environment is low. Monensin sodium from Elancoban® G200 has the potential to effectively control coccidiosis in chickens for fattening and chickens reared for laying. The FEEDAP Panel cannot conclude on the efficacy Elancoban® G200 as a coccidiostat for turkeys for fattening.

Comparative analyses of cellular physiological responses of non-target species to cypermethrin and its formulated product: Contribution to mode of action research.

Physiological responses of bacterial, fish, rat and human hepatoma cells to the technical cypermethrin (AS), cypermethrin-based plant protection product (PPP), and the major co-formulant (solvent) were compared. The endpoints included: bioluminescence, total protein content, activity of mitochondrial dehydrogenase and cytochrome P450 (CYP) enzymes CYP1A and CYP1B, and expression of several genes encoding different CYP enzyme isoforms. Toxicity of PPP was compared with the toxicity predicted using concentration addition model. Cypermethrin disturbs the activity of mitochondrial dehydrogenase. Induction of CYP1A1-, CYP1A2- and CYP1B1-associated activity was more pronounced in PPP than in cypermethrin treatment. The predominant biotransformation pathway of cypermethrin is related to Cyp3a1 induction. Deviations between observed and predicted toxicity of PPP indicate synergistic effects of cypermethrin and a solvent. In vitro cellular assays may serve as rapid pre-screening tool and provide for a good indication of mixture effects and prompt further in vivo testing of PPPs when really needed.

Effect-directed analysis (EDA) of Danube River water sample receiving untreated municipal wastewater from Novi Sad, Serbia.

The release of a multitude of pollutants from untreated municipal wastewater (UMWW) to surface waters may have adverse effects on aquatic wildlife including endocrine disruption. For effect-directed analysis (EDA), a Danube river water sample downstream of emission of UMWW in Novi Sad, Serbia was extracted on-site and after processing in the lab was subjected to reporter gene assays which revealed pronounced estrogenic (ERα), androgenic (AR) and oxidative stress response (OSR). The sample was fractionated with reversed-phase high performance liquid chromatography (RP-HPLC) collecting thirty fractions at two-minute intervals. Biological analysis identified 5 ERα- and 3 AR-active fractions while none of the fractions showed considerable activity with regards to OSR. It appeared that OSR of parent sample (PS) distributed over all fractions. Chemical analysis of active fractions by LC-MS/MS and LC-HRMS/MS found female reproductive hormones (estrone (E1), estradiol (E2), estriol (E3)) as cause of ERα activity while male reproductive hormones (testosterone, dihydrotestosterone (DHT)) and gestagens (progesterone and medroxyprogesterone) were active in the AR bioassay. Designed chemical mixtures in concentration ratios detected in the active fractions were tested with the bioassays. The identified chemicals quantitatively explained the observed bioactivity with no substantial contribution attributable to xenobiotics. In terms of bioanalytical equivalent concentrations (BEQs), detected chemicals explained 5-159% of ERα-active fraction's biological effect and 31-147% for AR-active fractions. Estradiol and dihydrotestosterone were the compounds dominating the most of the effect in this study. In summary, androgenic compounds were found to be as potent as estrogenic compounds while OSR was found to be the cumulative effect of the mixture of many compounds present in the sample rather than the mixture effect dominated by individual chemicals. The obtained results stress the importance of wastewater treatment plant (WWTP) to minimize the pollutant load from UMWW in order to reduce the risk of endocrine disruption to the aquatic life as well as to improve the status of receiving freshwater ecosystem.

Scientific opinion on pesticides in foods for infants and young children.

Following a request from the European Commission, the EFSA Panel on Plant Protection Products and their Residues (PPR Panel) prepared a scientific opinion to provide a comprehensive evaluation of pesticide residues in foods for infants and young children. In its approach to develop this scientific opinion, the EFSA PPR Panel took into account, among the others, (i) the relevant opinions of the Scientific Committee for Food setting a default maximum residue level (MRL) of 0.01 mg/kg for pesticide residues in foods for infants and young children; (ii) the recommendations provided by EFSA Scientific Committee in a guidance on risk assessment of substances present in food intended for infants below 16 weeks of age; (iii) the knowledge on organ/system development in infants and young children. For infants below 16 weeks of age, the EFSA PPR Panel concluded that pesticide residues at the default MRL of 0.01 mg/kg for food for infants and young children are not likely to result in an unacceptable exposure for active substances for which a health-based guidance value (HBGV) of 0.0026 mg/kg body weight (bw) per day or higher applies. Lower MRLs are recommended for active substances with HBGVs below this value. For infants above 16 weeks of age and young children, the established approach for setting HBGVs is considered appropriate. For infants below 16 weeks of age the approach may not be appropriate and the application of the EFSA guidance on risk assessment of substances present in food intended for infants below 16 weeks of age is recommended. The contribution of conventional food to the total exposure to pesticide residues is much higher than that from foods intended for infants and young children. Because of the increased intake of conventional food by young children, these have the highest exposure to pesticide residues, whereas infants 3-6 months of age generally have lower exposure. The impact of cumulative exposure to pesticide residues on infants and young children is not different from the general population and the EFSA cumulative risk assessment methodology is also applicable to these age groups. Residue definitions established under Regulation (EC) No 396/2005 are in general considered appropriate also for foods for infants and young children. However, based on a tier 1 analysis of the hydrolysis potential of pesticides simulating processing, the particular appropriateness of existing residue definitions for monitoring to cover processed food, both intended for infants and young children as well as conventional food, is questionable.

Scientific Opinion on the state of the art of Toxicokinetic/Toxicodynamic (TKTD) effect models for regulatory risk assessment of pesticides for aquatic organisms.

Following a request from EFSA, the Panel on Plant Protection Products and their Residues (PPR) developed an opinion on the state of the art of Toxicokinetic/Toxicodynamic (TKTD) models and their use in prospective environmental risk assessment (ERA) for pesticides and aquatic organisms. TKTD models are species- and compound-specific and can be used to predict (sub)lethal effects of pesticides under untested (time-variable) exposure conditions. Three different types of TKTD models are described, viz., (i) the 'General Unified Threshold models of Survival' (GUTS), (ii) those based on the Dynamic Energy Budget theory (DEBtox models), and (iii) models for primary producers. All these TKTD models follow the principle that the processes influencing internal exposure of an organism, (TK), are separated from the processes that lead to damage and effects/mortality (TD). GUTS models can be used to predict survival rate under untested exposure conditions. DEBtox models explore the effects on growth and reproduction of toxicants over time, even over the entire life cycle. TKTD model for primary producers and pesticides have been developed for algae, Lemna and Myriophyllum. For all TKTD model calibration, both toxicity data on standard test species and/or additional species can be used. For validation, substance and species-specific data sets from independent refined-exposure experiments are required. Based on the current state of the art (e.g. lack of documented and evaluated examples), the DEBtox modelling approach is currently limited to research applications. However, its great potential for future use in prospective ERA for pesticides is recognised. The GUTS model and the Lemna model are considered ready to be used in risk assessment.

Scientific Opinion about the Guidance of the Chemical Regulation Directorate (UK) on how aged sorption studies for pesticides should be conducted, analysed and used in regulatory assessments.

The EFSA Panel on Plant Protection Products and their Residues reviewed the guidance on how aged sorption studies for pesticides should be conducted, analysed and used in regulatory assessment. The inclusion of aged sorption is a higher tier in the groundwater leaching assessment. The Panel based its review on a test with three substances taken from a data set provided by the European Crop Protection Association. Particular points of attention were the quality of the data provided, the proposed fitting procedure of aged sorption experiments and the proposed method for combining results obtained from aged sorption studies and lower-tier studies on degradation and adsorption. Aged sorption was a relevant process in all cases studied. The test revealed that the guidance could generally be well applied and resulted in robust and plausible results. The Panel considers the guidance suitable for use in the groundwater leaching assessment after the recommendations in this Scientific Opinion have been implemented, with the exception of the use of field data to derive aged sorption parameters. The Panel noted that the draft guidance could only be used by experienced users because there is no software tool that fully supports the work flow in the guidance document. It is therefore recommended that a user-friendly software tool be developed. Aged sorption lowered the predicted concentration in groundwater. However, because aged sorption experiments may be conducted in different soils than lower-tier degradation and adsorption experiments, it cannot be guaranteed that the higher tier predicts lower concentrations than the lower tier, while lower tiers should be more conservative than higher tiers. To mitigate this problem, the Panel recommends using all available higher- and lower-tier data in the leaching assessment. The Panel further recommends that aged sorption parameters for metabolites be derived only from metabolite-dosed studies. The formation fraction can be derived from parent-dosed degradation studies, provided that the parent and metabolite are fitted with the best-fit model, which is the double first-order in parallel model in the case of aged sorption.

Scientific Opinion of the PPR Panel on the follow-up of the findings of the External Scientific Report 'Literature review of epidemiological studies linking exposure to pesticides and health effects'.

In 2013, EFSA published a comprehensive systematic review of epidemiological studies published from 2006 to 2012 investigating the association between pesticide exposure and many health outcomes. Despite the considerable amount of epidemiological information available, the quality of much of this evidence was rather low and many limitations likely affect the results so firm conclusions cannot be drawn. Studies that do not meet the 'recognised standards' mentioned in the Regulation (EU) No 1107/2009 are thus not suited for risk assessment. In this Scientific Opinion, the EFSA Panel on Plant Protection Products and their residues (PPR Panel) was requested to assess the methodological limitations of pesticide epidemiology studies and found that poor exposure characterisation primarily defined the major limitation. Frequent use of case-control studies as opposed to prospective studies was considered another limitation. Inadequate definition or deficiencies in health outcomes need to be avoided and reporting of findings could be improved in some cases. The PPR Panel proposed recommendations on how to improve the quality and reliability of pesticide epidemiology studies to overcome these limitations and to facilitate an appropriate use for risk assessment. The Panel recommended the conduct of systematic reviews and meta-analysis, where appropriate, of pesticide observational studies as useful methodology to understand the potential hazards of pesticides, exposure scenarios and methods for assessing exposure, exposure-response characterisation and risk characterisation. Finally, the PPR Panel proposed a methodological approach to integrate and weight multiple lines of evidence, including epidemiological data, for pesticide risk assessment. Biological plausibility can contribute to establishing causation.

Investigation into experimental toxicological properties of plant protection products having a potential link to Parkinson's disease and childhood leukaemia.

In 2013, EFSA published a literature review on epidemiological studies linking exposure to pesticides and human health outcome. As a follow up, the EFSA Panel on Plant Protection Products and their residues (PPR Panel) was requested to investigate the plausible involvement of pesticide exposure as a risk factor for Parkinson's disease (PD) and childhood leukaemia (CHL). A systematic literature review on PD and CHL and mode of actions for pesticides was published by EFSA in 2016 and used as background documentation. The Panel used the Adverse Outcome Pathway (AOP) conceptual framework to define the biological plausibility in relation to epidemiological studies by means of identification of specific symptoms of the diseases as AO. The AOP combines multiple information and provides knowledge of biological pathways, highlights species differences and similarities, identifies research needs and supports regulatory decisions. In this context, the AOP approach could help in organising the available experimental knowledge to assess biological plausibility by describing the link between a molecular initiating event (MIE) and the AO through a series of biologically plausible and essential key events (KEs). As the AOP is chemically agnostic, tool chemical compounds were selected to empirically support the response and temporal concordance of the key event relationships (KERs). Three qualitative and one putative AOP were developed by the Panel using the results obtained. The Panel supports the use of the AOP framework to scientifically and transparently explore the biological plausibility of the association between pesticide exposure and human health outcomes, identify data gaps, define a tailored testing strategy and suggests an AOP's informed Integrated Approach for Testing and Assessment (IATA).

Impact of untreated wastewater on a major European river evaluated with a combination of in vitro bioassays and chemical analysis.

Complex mixtures of micropollutants, including pesticides, pharmaceuticals and industrial chemicals emitted by wastewater effluents to European rivers may compromise the quality of these water resources and may pose a risk to ecosystem health and abstraction of drinking water. In the present study, an integrated analytical and bioanalytical approach was applied to investigate the impact of untreated wastewater effluents from the city of Novi Sad, Serbia, into the River Danube. The study was based on three on-site large volume solid phase extracted water samples collected upstream and downstream of the untreated wastewater discharge. Chemical screening with liquid chromatography high resolution mass spectrometry (LC-HRMS) was applied together with a battery of in vitro cell-based bioassays covering important steps of the cellular toxicity pathway to evaluate effects on the activation of metabolism (arylhydrocarbon receptor AhR, peroxisome proliferator activated receptor gamma PPARγ), specific modes of action (estrogen receptor ERα, androgen receptor AR) and adaptive stress responses (oxidative stress, inflammation). Increased effects, significantly changed contamination patterns and higher chemical concentrations were observed downstream of the wastewater discharge. A mass balance approach showed that enhanced endocrine disruption was in good agreement with concentrations of detected hormones, while only a smaller fraction of the effects on xenobiotic metabolism (<1%) and adaptive stress responses (0-12%) could be explained by the detected chemicals. The chemical and effects patterns observed upstream of the discharge point were fairly re-established at about 7 km downstream, demonstrating the enormous dilution capacity of this large river.

Longitudinal profile of the genotoxic potential of the River Danube on erythrocytes of wild common bleak (Alburnus alburnus) assessed using the comet and micronucleus assay.

The Joint Danube Survey 3 (JDS3; the biggest river expedition in 2013) had offered the unique opportunity for a large-scale monitoring approach for biomarker response in feral fish collected along a Danube stretch from Kehlheim (DE) to Sulina (RO). The advantage of genotoxicity as a marker for pollution exposure in fish is the early detection of possible long-term effects such as cancer. Therefore, genotoxicity was in the focus of the biomarker investigations in fish during the expedition. Blood samples of common bleak (Alburnus alburnus) for the investigation of the micronucleus frequency and comet tail intensity of fragmented DNA material in erythrocytes were collected at 18 and 12 sampling sites, respectively. For 9 sampling sites same samples were used to compare the in-situ data for the comparable genotoxic endpoint in the micronucleus (MN) and comet assay (CM). The data of both in-situ assays showed a significant correlation, indicating the strength and comparability of the data sets. Significant variation in DNA damage in fish along the longitudinal profile of the Danube was demonstrated for both assays compared to reference sites. The results suggest that DNA damage in erythrocytes of fish was mainly affected by wastewater of highly populated regions. No linkage between the results and the general health/dietary status of the fish were revealed, whereas correlation with some genotoxicity drivers in the water phase, suspended particulate matter and sediments could be demonstrated.

Differential expression of CYP1A1 and CYP1A2 genes in H4IIE rat hepatoma cells exposed to TCDD and PAHs.

Rat hepatoma cells H4IIE were treated by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and polycyclic aromatic hydrocarbons (PAHs) (dibenz(a,h)anthracene, benzo(a)pyrene, benz(a)anthracene, chrysene), low-concentration mixtures of PAHs and TCDD, and environmental mixtures contaminated by PAHs and their derivatives. Expression of the gene battery comprising cytochrome P450 Cyp1a1, Cyp1a2, Cyp1b1, and glutathione-s-transferase Gsta2 and Gstp was investigated using quantitative real time polymerase chain reaction (qRT-PCR) analysis. The results revealed that TCDD induce Cyp1a1>Cyp1a2>Cyp1b1, while PAHs and PAH-containing environmental mixtures induce Cyp1a2>Cyp1a1>Cyp1b1 gene expression pattern. While low-concentration mixtures elicited a more pronounced response in comparison to single treatments, the typical gene expression patterns were not observed. In all samples, Gsta2 was predominantly expressed relative to Gstp. These findings indicate that differential Cyp1a1 and Cyp1a2 expression in the H4IIE cells might be used for detection of PAHs in highly contaminated environmental mixtures, but not in low-concentration mixtures of these compounds.

Effect-directed analysis of contaminated sediment from the wastewater canal in Pancevo industrial area, Serbia.

Wastewater canal (WWC) in Pancevo industrial area in Serbia, whose main environmental receptor is the River Danube, is a well known hot-spot of contamination. WWC sediments have been assessed by UNEP based on chemical target analysis. However, integrative biological data on exposure to hazardous compounds are only provided by the present study which aims at evaluating whether the monitored compounds sufficiently reflect potential hazards and to suggest additional compounds to include in monitoring and hazard assessment by applying effect-directed analysis (EDA) based on arylhydrocarbon receptor-mediated activity and cytotoxicity. Multistep NP-HPLC fractionation provided 18 fractions co-eluting with polychlorinated biphenyls (PCBs), polychlorinated dibenzo-p-dioxins (PCDDs), polycyclic aromatic hydrocarbons (PAHs) and more polar compounds. PAHs fractions exhibited great potencies to induce ethoxyresorufin-o-deethylase (EROD) in H4IIE rat hepatoma cell line expressed as 2,3,7,8-tetrachlorodibenzo-p-dioxin equivalents (TCDD-EQ) (0.1-34.6 x 10(3) pg g(-1)dry weight). Chemical analysis of the most active fractions revealed great concentrations of PAHs (up to 292 x 10(2)ngg(-1) sediment equivalents (SEQ)), methylated PAHs (up to 900 x 10(2) ng g(-1) SEQ), and other alkyl-substituted PAHs. Only minor portions of biologically derived TCDD-EQs could be attributed to monitored PAHs with known relative potencies (REPs). We hypothesize that a major part of the activity is due to non-monitored alkylated and heterocyclic PAHs. Results of the cell cytotoxicity/proliferation assay on H4IIE cell line suggest the presence of sediment pollutants with pronounced potency to disturb cell growth.

Detection of dioxin-like contaminants in soil from the area of oil refineries in Vojvodina region of Serbia.

In this study level of soil contamination by polychlorinated biphenyls (PCBs) and polycyclic aromatic hydrocarbons (PAHs) in two oil refineries in Vojvodina region of Serbia was assessed using combined bio/chemical approach. Toxicity of the samples, determined by microEROD analysis, could not be exclusively attributed to the content of measured PCBs and PAHs, but also to the presence of unknown dioxin-like compounds (DLC), and/or positive interactions among similarly acting chemicals. The results proved that biotests, when applied in ecotoxicological assessments, should be used either as a screening tool or initial step in effect-directed analyses.

Rituximab maintenance improves clinical outcome of relapsed/resistant follicular non-Hodgkin lymphoma in patients both with and without rituximab during induction: results of a prospective randomized phase 3 intergroup trial.

We evaluated the role of rituximab (R) both in remission induction and maintenance treatment of relapsed/resistant follicular lymphoma (FL). A total of 465 patients were randomized to induction with 6 cycles of cyclophosphamide, doxorubicin, vincristine, and prednisone (CHOP) (every 3 weeks) or R-CHOP (R: 375 mg/m(2) intravenously, day 1). Those in complete remission (CR) or partial remission (PR) were randomized to maintenance with R (375 mg/m(2) intravenously once every 3 months for a maximum of 2 years) or observation. R-CHOP induction yielded an increased overall response rate (CHOP, 72.3%; R-CHOP, 85.1%; P < .001) and CR rate (CHOP, 15.6%; R-CHOP, 29.5%; P < .001). Median progression-free survival (PFS) from first randomization was 20.2 months after CHOP versus 33.1 months after R-CHOP (hazard ratio [HR], 0.65; P < .001). Rituximab maintenance yielded a median PFS from second randomization of 51.5 months versus 14.9 months with observation (HR, 0.40; P < .001). Improved PFS was found both after induction with CHOP (HR, 0.30; P < .001) and R-CHOP (HR, 0.54; P = .004). R maintenance also improved overall survival from second randomization: 85% at 3 years versus 77% with observation (HR, 0.52; P = .011). This is the first trial showing that in relapsed/resistant FL rituximab maintenance considerably improves PFS not only after CHOP but also after R-CHOP induction.

Phase III intergroup study of fludarabine phosphate compared with cyclophosphamide, vincristine, and prednisone chemotherapy in newly diagnosed patients with stage III and IV low-grade malignant Non-Hodgkin's lymphoma.

PURPOSE: To compare the efficacy and safety of fludarabine phosphate with cyclophosphamide, vincristine, and prednisone (CVP) in 381 previously untreated, advanced-stage, low-grade (lg) non-Hodgkin's lymphoma (NHL) patients in a phase III, multicenter study. PATIENTS AND METHODS: Between 1993 and 1997, patients were randomly assigned to treatment with either fludarabine (25 mg/m2 intravenously [IV] daily for 5 days every 4 weeks) or CVP (cyclophosphamide 750 mg/m2 IV on day 1; vincristine, 1.4 mg/m2 IV on day 1; and prednisone, 40 mg/m2 orally on days 1 through 5 every 4 weeks). Results Overall response (OR) rates were significantly improved in the fludarabine arm versus the CVP arm, both for the intent-to-treat (ITT) population and assessable patients (P < .001). Complete response (CR) rates in the ITT population were also higher after fludarabine treatment. The CR rate was 38.6% for fludarabine compared with 15.0% for CVP. There were no statistically significant differences in time to progression (TTP), time to treatment failure (TTF), and overall survival (OS) between treatment groups. WHO grades 3 and 4 hematologic adverse events were more common in the fludarabine arm. However, concerning the higher incidence of granulocytopenia, this did not translate to more infections in fludarabine-treated patients. CONCLUSION: Newly diagnosed lgNHL patients who received fludarabine achieved higher OR and CR rates compared with CVP-treated patients. No differences in TTP, TTF, and OS were noted. Fludarabine is a highly active single agent in lgNHL. Combination therapies incorporating fludarabine are now being further evaluated as first-line therapy in follicular NHL.

High-dose methotrexate-based chemotherapy followed by consolidating radiotherapy in non-AIDS-related primary central nervous system lymphoma: European Organization for Research and Treatment of Cancer Lymphoma Group Phase II Trial 20962.

PURPOSE: To confirm the feasibility and estimate the efficacy of methotrexate (MTX), teniposide, carmustine, and methylprednisolone (MBVP) chemotherapy combined with radiotherapy (RT) for patients with non-AIDS-related primary CNS lymphoma (PCNSL) treated in a multicenter setting. PATIENTS AND METHODS: Treatment consisted of two cycles of MBVP (MTX 3 g/m2 days 1 and 15, teniposide 100 mg/m2 days 2 and 3, carmustine 100 mg/m2 day 4, methylprednisolone 60 mg/m2 days 1 to 5, and two intrathecal injections of MTX 15 mg, cytarabine 40 mg, and hydrocortisone 25 mg) followed by 40 Gy of RT. Primary end points were response and safety of this regimen. RESULTS: Twelve centers included 52 patients who were all analyzed on an intent-to-treat basis. Median follow-up of all patients was 27 months. One patient progressed and died before treatment, and five patients died during treatment. Four patients received RT after one cycle of chemotherapy, and 42 patients completed the entire treatment. Hematologic grade 3 and 4 toxicity was seen in 78% of patients for leukocytes and 24% of patients for platelets. The overall response rate of all 52 patients was 81%. Two patients who did not fulfill the criteria of objective response survived more than 1 year; one of them is still alive without disease. Eighteen patients died; 11 deaths were a result of tumor, five were probably treatment-related, one was caused by late leukoencephalopathy, and one was a result of intercurrent disease. Median estimated overall survival was 46 months. CONCLUSION: MBVP followed by RT for PCNSL has a high response rate. However, the 10% toxic death rate during treatment in a multicenter setting underlines the need for highly specialized care.