Post-induction Phase Research Articles

Dual effects of a high-protein diet on DSS-treated mice during colitis resolution phase.

The impact of the dietary protein level on the process of colonic mucosal inflammation and subsequent recovery remains largely unknown. In this study, we fed DSS-treated mice with either a normoproteic (NP) or a high-protein (HP) isocaloric diet from the beginning of the 5-day dextran sulfate sodium (DSS) treatment to 14 days later. Measurements of colitis indicators (colon weight:length ratio, myeloperoxidase activity, cytokine expressions) showed a similar level of colonic inflammation in both DSS groups during the colitis induction phase. However, during the colitis resolution phase, inflammation intensity was higher in the DSS-HP group than in the DSS-NP group as evidenced by higher inflammatory score and body weight loss. This coincided with a higher mortality rate. In surviving animals, an increase in colonic crypt height associated with a higher number of colon epithelial cells per crypt, and TGF-β3 content was observed in the DSS-HP vs. DSS-NP group. Moreover, colonic expression patterns of tight junction proteins and E-cadherin were also different according to the diet. Altogether, our results indicate that the HP diet, when given during both the induction and resolution periods of DSS-induced colitis, showed deleterious effects during the post-induction phase. However, HP diet ingestion was also associated with morphological and biochemical differences compatible with higher colonic epithelium restoration in surviving animals, indicating an effect of the dietary protein level on colonic crypt repair after acute inflammation. These data highlight the potential impact of the dietary protein amount during the colitis course.

A game for emotional regulation in adolescents: The (body) interface device matters

The aim of this study is to evaluate the role of the type of interface device in the efficacy of a serious game that teaches emotional regulation (ER) strategies in a non-clinical sample of adolescents. We conducted a between-participants experiment in which participants (N = 61) played a frustration induction game, and then an ER game (a breathing strategy game), using one of three types of devices (computer, smartphone, and RGB-D camera). Frustration mood and perceived arousal were the main variables measured. Results revealed a significant interaction between moment (pre-induction phase, post-induction phase, and regulation phase) and the type of interface device used in the frustration mood scores. In participants who used the computer and smartphone, frustration increased after the induction phase and decreased after the regulation phase. However, for participants who used the RGB-D camera, frustration decreased significantly after the induction phase, and this change was maintained after the regulation phase. Changes in arousal were similar with the three devices. This study highlights that the type of interface device (and specifically, the participation of the body) is a crucial variable in the efficacy of serious games affecting users’ emotional experience.

Effectiveness and safety of post-induction phase bevacizumab treatment for patients with non-small-cell lung cancer: results from the ARIES observational cohort study.

Data from randomized, controlled trials suggest that post-induction phase (IP) treatment with bevacizumab may benefit patients with advanced non-small-cell lung cancer (NSCLC). Real-world clinical practice, however, can involve variable use and patterns of treatment in broader patient populations. To assess the effect of bevacizumab on post-IP overall survival (OS) following IP chemotherapy + bevacizumab, analyses were conducted in patients enrolled in the Avastin(®) Registry--Investigation of Effectiveness and Safety (ARIES) observational cohort study (OCS) who received post-IP bevacizumab. ARIES was a large, prospective OCS of patients who received chemotherapy in combination with bevacizumab for the first-line treatment of NSCLC. This unplanned, post hoc analysis included patients who received chemotherapy and bevacizumab and who did not have progressive disease through the completion of IP treatment. A dichotomous analysis compared outcomes in patients who did and did not receive bevacizumab before a landmark date of day 30 post IP. A cumulative exposure analysis used a time-dependent Cox regression model to assess the effect of cumulative post-IP bevacizumab exposure on post-IP OS. In the dichotomous analysis, the duration of post-IP OS was significantly longer in patients who received post-IP bevacizumab; median post-IP OS was 15.6 vs. 11.3 months, respectively (hazard ratio [HR] = 0.80; 95 % confidence interval 0.71-0.91; P < 0.001). The cumulative exposure analysis observed that each additional cycle of cumulative bevacizumab exposure decreased the HR for post-IP OS by 2.7 %, on average. In conclusion, post-IP bevacizumab exposure was associated with improved post-IP OS in patients with advanced NSCLC who were enrolled in the ARIES OCS.

Treatment of elderly patients with acute lymphoblastic leukaemia using a paediatric‐based protocol

All newly diagnosed patients aged 60-79 years with acute lymphoblastic leukaemia (ALL) receiving induction chemotherapy with a modified paediatric-based regimen over a 7-year period were retrospectively analysed (n = 51, median age 65 years). The treatment regimen consisted of induction, central nervous system prophylaxis, seven cycles of intensification and 24 cycles of maintenance. BCR-ABL1 negative patients received weekly asparaginase during intensification, while BCR-ABL1+ patients received daily imatinib. Post-remission therapy was given in an outpatient setting. The complete response rate was 75%, with an induction mortality of 20%; 6% of patients had resistant disease. 37% of patients who achieved a complete remission relapsed. The estimated 5-year overall survival was 40% for BCR-ABL1 negative and 47% for BCR-ABL1+ patients (P = not significant); the 5-year disease-free survival was 57% and 39%, respectively (P = NS). The post-induction phase was generally well tolerated, with 81% able to complete the intensification phase and proceed to maintenance. In conclusion, administration of this modified paediatric-based protocol is feasible and active for elderly patients with ALL. Survival is superior to most previously reported series in this age group, but remains worse compared to younger patients. Further improvement of the toxicity profile, particularly during induction, is required to improve outcomes.

1278P - Cumulative Exposure (EXP) to Bevacizumab (BV) Maintenance after Induction Therapy and Survival in Advanced Non-Small Cell Lung Cancer (NSCLC): A Time-Dependent Analysis from the Sail (MO19390) Study

ABSTRACT Background Exploratory analyses from randomized and observational studies have shown that continuation BV until progressive disease (PD) is associated with prolonged overall survival (OS) in advanced NSCLC. We evaluated the correlation between cumulative exp to post-induction phase (post-IP) BV and OS in patients (pts) with NSCLC. Methods The multinational phase 4 SAiL study enrolled advanced NSCLC pts receiving BV with 1st-line chemotherapy (CT). Pts who began BV and CT simultaneously and did not have PD after completing 12–18 weeks (4–6 cycles) of CT were included for analysis. OS was measured from the end of each pt's BV + CT IP. BV exp, over follow-up, was defined as the cumulative days of post-IP BV treatment (tx). A time-dependent Cox regression model was fitted to assess the effect of cumulative post-IP BV exp on OS, while controlling for potential time-dependent and -fixed confounders. A landmark sensitivity analysis compared OS in pts in the analysis population who did and did not continue BV after IP. Results Of 2212 pts in SAiL, 1625 NSCLC pts were alive and without PD through IP tx. Baseline characteristics (n = 1625) were: 58% male, 13% ≥70 y, 32% never smokers, and 4% ECOG PS ≥2. 1047 (64%) pts received post-IP BV. Baseline and end-of-IP characteristics were generally similar between pts receiving post-IP BV or no post-IP BV within the landmark period. Across follow-up, the hazard ratios (HRs) for OS decreased by ∼7% (range, 5%–9%) with each additional 21-day interval of cumulative exp (Table). Landmark sensitivity analyses also support that post-IP BV was associated with longer OS (post-IP BV vs no post-IP BV; HR, 0.74; 95% CI, 0.64–0.85). Conclusions These data from SAiL along with previously presented data from ARIES suggest that cumulative exp to post-IP BV is associated with incremental increases in OS for NSCLC pts. Cumulative post-IP BV cycles a No. of pts who received cumulative cycles of post-IP BV continuously b HR (95% confidence interval) for OS 1 1006 0.91 (0.89–0.93) 2 884 0.82 (0.79–0.86) 3 758 0.75 (0.70–0.80) 4 643 0.68 (0.62–0.74) 5 530 0.62 (0.55–0.69) 6 447 0.56 (0.49–0.64) 7 370 0.51 (0.44–0.59) 8 315 0.46 (0.39–0.55) a A cycle is approximately 21 days of post-IP cumulative exp. b Eg, n = 530 pts were continuously dosed through day 105 (cycle 5) after IP. Disclosure N. Thatcher: Dr. Thatcher is a member of the Roche advisory board and has received speaker fees from Roche. P. Garrido Lopez: Dr. Garrido Lopez is a member of the Roche advisory board. N. Pavlakis: Dr. Pavlakis has received speaker honoraria and travel grants from Roche, and has participated in Roche Advisory Boards. J. Laskin: Dr. Laskin is a member of the Canadian National Ad Board for lung cancer for Astra Zeneca, BI, and Eli Lilly and has received research funding from Roche, Boehringer-Ingelheim, and Eli Lilly. E. Dansin: Dr. Dansin is a member of the Roche advisory board. F. Griesinger: Dr. Griesinger is a member of national and international advisory boards of Roche and has received honoraria for presentations at meetings. L. Leon: Dr. Leon is an employee of and holds stock options in Roche/Genentech. D. Dalal: Dr. Dalal is an employee of and holds stock options in Roche/Genentech. P. Perez-Moreno: Dr. Perez-Moreno is an employee of and holds stock options in F. Hoffman LaRoche. L. Crino: Dr. Crino received honorarium from Roche as a speaker in a scientific symposium.

Generation of diploid Pichia pastoris strains by mating and their application for recombinant protein production

BackgroundYeast mating provides an efficient means for strain and library construction. However, biotechnological applications of mating in the methylotrophic yeast Pichia pastoris have been hampered because of concerns about strain stability of P. pastoris diploids. The aim of the study reported here is to investigate heterologous protein expression in diploid P. pastoris strains and to evaluate diploid strain stability using high cell density fermentation processes.ResultsBy using a monoclonal antibody as a target protein, we demonstrate that recombinant protein production in both wild-type and glycoengineered P. pastoris diploids is stable and efficient during a nutrient rich shake flask cultivation. When diploid strains were cultivated under bioreactor conditions, sporulation was observed. Nevertheless, both wild-type and glycoengineered P. pastoris diploids showed robust productivity and secreted recombinant antibody of high quality. Specifically, the yeast culture maintained a diploid state for 240 h post-induction phase while protein titer and N-linked glycosylation profiles were comparable to that of a haploid strain expressing the same antibody. As an application of mating, we also constructed an antibody display library and used mating to generate novel full-length antibody sequences.ConclusionsTo the best of our knowledge, this study reports for the first time a comprehensive characterization of recombinant protein expression and fermentation using diploid P. pastoris strains. Data presented here support the use of mating for various applications including strain consolidation, variable-region glycosylation antibody display library, and process optimization.

Overproduction of alkaline polygalacturonate lyase in recombinant Escherichia coli by a two-stage glycerol feeding approach

This work aims to achieve the overproduction of alkaline polygalacturonate lyase (PGL) with recombinant Escherichia coli by a two-stage glycerol feeding approach. First, the PGL coding gene from Bacillus subtilis WSHB04–02 was expressed in E. coli BL21 (DE3) under the strong inducible T7 promoter of the pET20b (+) vector. And then the influence of media composition, induction temperature, and inducer isopropyl β-d-1-thiogalactopyranoside (IPTG) concentration on cell growth and PGL production was investigated. Finally, a two-stage glycerol feeding strategy was proposed and applied in a 3-L fermenter, where cultivation was conducted at a controlled specific growth rate (μset=0.2) during pre-induction phase, followed by a constant glycerol feeding rate of 12mlh−1 at post-induction phase. The total PGL yield reached 371.86UmL−1, which is the highest PGL production by recombinant E. coli expression system.

KPNA6 (Importin α7)-Mediated Nuclear Import of Keap1 Represses the Nrf2-Dependent Antioxidant Response

The transcription factor Nrf2 has emerged as a master regulator of cellular redox homeostasis. As an adaptive response to oxidative stress, Nrf2 activates the transcription of a battery of genes encoding antioxidants, detoxification enzymes, and xenobiotic transporters by binding the cis-antioxidant response element in the promoter regions of genes. The magnitude and duration of inducible Nrf2 signaling is delicately controlled at multiple levels by Keap1, which targets Nrf2 for redox-sensitive ubiquitin-mediated degradation in the cytoplasm and exports Nrf2 from the nucleus. However, it is not clear how Keap1 gains access to the nucleus. In this study, we show that Keap1 is constantly shuttling between the nucleus and the cytoplasm under physiological conditions. The nuclear import of Keap1 requires its C-terminal Kelch domain and is independent of Nrf1 and Nrf2. We have determined that importin α7, also known as karyopherin α6 (KPNA6), directly interacts with the Kelch domain of Keap1. Overexpression of KPNA6 facilitates Keap1 nuclear import and attenuates Nrf2 signaling, whereas knockdown of KPNA6 slows down Keap1 nuclear import and enhances the Nrf2-mediated adaptive response induced by oxidative stress. Furthermore, KPNA6 accelerates the clearance of Nrf2 protein from the nucleus during the postinduction phase, therefore promoting restoration of the Nrf2 protein to basal levels. These findings demonstrate that KPNA6-mediated Keap1 nuclear import plays an essential role in modulating the Nrf2-dependent antioxidant response and maintaining cellular redox homeostasis.

Effects of acetic acid and its assimilation in fed-batch cultures of recombinant Escherichia coli containing human-like collagen cDNA

The primary processing problem in recombinant Escherichia coli fermentation is the production of acetic acid, which can inhibit both cell growth and recombinant protein production. The ability of E. coli to assimilate acetate permits it to solve this problem in a rather creative manner. In this study, the effects of acetic acid assimilation through a glucose starvation period at different cell growth phases were investigated in fed-batch cultures of recombinant E. coli. Experimental results showed that the human-like collagen (HLC) production could be improved by introducing glucose starvation at the end of batch culture and pre-induction phase, while the glucose starvation at the induction phase resulted in a poor HLC productivity. The acetic acid assimilation was observed during all the glucose starvation periods. In addition, a systematic study for evaluating the effects of acetic acid was carried out by adding acetate into culture media at different cell growth phases and then employing a glucose starvation after several hours. It was found that obvious acetate inhibition on cell growth occurred in the batch culture phases while its inhibitory effect on HLC expression occurred only in the post-induction phase. The longer the elevated acetic acid concentration maintained, the stronger the inhibitory effects were. These results are of significance for optimizing and scaling-up fermentation processes.

Cellular response to accumulation of recombinant proteins in the E. coli inner membrane: Implications for proteolysis and productivity of the secretory expression system

Several literature reports have indicated adverse effects on host cell growth and physiology due to secretory expression of recombinant proteins. In this work, extensive proteolysis of recombinant streptokinase was observed in the secretory expression system, along with severe growth impairment and reduced productivity as compared to intracellular expression in Escherichia coli. These phenomena correlated well with the accumulation of the secretory recombinant protein in the inner membrane and a corresponding up-regulation of an inner-membrane protease (probably ClpX). This protease was found to be activated only in the post-induction phase of the secretory expression system. A reduction in the cultivation temperature led to a significant decrease in the accumulation level of recombinant streptokinase in the inner membrane and a concomitant decrease in the inner-membrane protease activity. This resulted in a considerable enhancement in the overall productivity of the secretory expression system. The results demonstrate that the productivity of the secretory expression system is controlled by the accumulation level of the recombinant protein in the inner membrane. Beyond a particular accumulation level the cellular response triggered in the form impaired growth and enhanced proteolysis significantly affects the productivity of the system. In order to reduce the accumulation in the inner membrane, the synthesis rate needs to be tuned to the protein-specific translocation efficiency of the cell.

Beneficial properties of melatonin in an experimental model of pancreatic cancer

Pancreatic cancer is a major health problem because of the aggressiveness of the disease and the lack of effective systemic therapies. Melatonin has antioxidant activity and prevents experimental genotoxicity. However, the effect of melatonin in pancreatic cancer has not been tested. Pancreatic carcinogenesis was induced by N-nitrosobis (2-oxopropyl)amine (BOP) in Syrian hamsters. Melatonin was administered during the BOP-induction phase (12 wk) and/or following the postinduction phase (12 wk). Different parameters of oxidative stress including lipid peroxides (LPO) and antioxidants (superoxide dismutase, catalase, reduced glutathione and glutathione peroxidase) were determined in pancreatic tissue. Also, the presence of atypical hyperplasia (AH), well and moderately differentiated adenomacarcinoma (ADC-WD and ADC-MD, respectively) were studied. The administration of BOP induced an intense oxidative stress and ADC induction in the pancreas. The administration of melatonin during the induction or postinduction phase reduced LPO and improved the antioxidant status, as well as drastically reducing the presence of ADC but some AH remained. In conclusion, treatment with melatonin reduced oxidative damage and cancer nodules induced by BOP in the pancreas.

Effects of post-induction feed strategies on secretory production of recombinant streptokinase in Escherichia coli

The effects of post-induction nutrient feed rates, on recombinant streptokinase production in fed-batch processes, were investigated using various feed profiles. Recombinant streptokinase was produced using a secretory expression system and was induced by a temperature up-shift, using a temperature-sensitive λ PL promoter. The specific growth rates decreased sharply upon induction of recombinant protein expression, thus necessitating a variable feed strategy in the post-induction phase. The various feed profiles employed in the post-induction phase included constant feed rates, linearly increasing feed rate and exponentially varying feed rates. Significant differences were obtained in the specific activity of streptokinase produced in these fed-batch processes. A maximum activity per unit biomass of 4.96 × 10 6 and 4.43 × 10 6 IU/g DCW was achieved for exponentially decreasing feed and linearly increasing feed, respectively. The decrease in specific growth rate during the post-induction phase was also less pronounced in these cases in comparison to other fed-batch experiments. It was observed that streptokinase productivity is governed by the nutrient feed rate per unit biomass at a critical juncture after induction. The highest activity per unit biomass was obtained when the nutrient feed rate per unit biomass was around 0.7–0.8 g glucose (g DCW) −1 h −1, between 2 and 4 h after induction.

Relapsing acute myeloid leukaemia manifesting as uveitis with hypopyon

A 5-year-old boy presented with a history of recurrent cough, lethargy and weight loss. On examination he had generalised lymphadenopathy, hepatosplenomegaly and bilateral pleural effusions. His full blood count at diagnosis was: haemoglobin 98 g/l, white cell count 138 × 109/l and platelet count 43 × 109/l. Bone marrow examination confirmed acute myeloid leukaemia and a cerebrospinal fluid cytospin was clear. Cytogenetic analysis showed 46,XY,der (1) t (1;1) (p36;q2?3) and trisomy 8. Pleural fluid aspirate was consistent with leukaemic infiltration. He was treated with chemotherapy, according to the protocol of the Acute Myeloid Leukaemia 15 trial, into which he was entered. He achieved complete morphological and cytogenetic remission post-induction phase. He re-presented with an 8-day history of left eye pain and low-grade temperature of 37·5°C. His full blood count was as follows: haemoglobin 119 g/l, white cell count 9·2 × 109/l and platelet count 268 × 109/l. He was prescribed neomycin eye ointment and oral cephalexin. He continued to deteriorate with worsening left eye symptoms, photophobia, lethargy and feeling generally unwell. He was reviewed by an ophthalmologist and was found to have an inflamed sclerae and marked inflammatory activity at the anterior chamber with a 2–3 mm hypopyon. He was re-examined under a general anaesthetic and was found to have corneal oedema of the left eye with a markedly thickened pale iris with dilated circumferential vessels; a clinical diagnosis of leukaemic infiltration of the iris was made (right and left panel). A diagnostic tap was felt to be unnecessary. A bone marrow aspirate and lumbar puncture were performed and confirmed bone marrow and central nervous system relapse. He was commenced on FLAG-I (fludarabine, cytarabine, idarubicin, granulocyte colony-stimulating factor) and weekly intrathecal chemotherapy. He had a further course of chemotherapy and underwent an allogeneic bone marrow transplantation. He engrafted but died <6 months later with extramedullary disease. Leukaemic hypopyon, as seen in this patient, is a rare manifestation of acute myeloid leukaemia.

Occurrence of thrombotic events in acute promyelocytic leukemia correlates with consistent immunophenotypic and molecular features

Although the occurrence of thrombosis in acute promyelocytic leukemia (APL) has been reported during retinoic acid treatment, no studies carried out in large clinical cohorts have specifically addressed this issue. We analyzed 124 APL patients treated with the all-trans retinoic acid and idarubicin protocol and compared clinico-biologic characteristics of 11 patients who developed thrombosis with those of 113 patients who had no thrombosis. In seven patients, the events were recorded during induction, whereas in four patients deep vein thrombosis occurred in the post-induction phase. Comparison of clinico-biological characteristics of patients with and without thrombosis revealed in the former group higher median white blood cell (WBC) count (17 x 10(9)/l, range 1.2-56, P=0.002), prevalence of the bcr3 transcript type (72 vs 48%, P=0.01), of FLT3-ITD (64 vs 28%, P=0.02), CD2 (P=0.0001) and CD15 (P=0.01) expression. No correlation was found with sex, age, French-American-British subtype, all-trans-retinoic acid syndrome or with thrombophilic state that was investigated in 5/11 patients. Our findings suggest that, in APL patients consistent biologic features of leukemia cells may predict increased risk of developing thrombosis.

The Value of Molecular Monitoring for Residual Disease (MRD) in Early Morphological Remitters among Adults Diagnosed with B Cell ALL and Treated According to the MRC UKALL12 Protocol.

Molecular monitoring of minimal residual disease (MRD) has become an independent and prognostically significant parameter in assessing outcome in adult and childhood acute lymphoblastic leukaemia (ALL). The aim of our study was to evaluate the impact of MRD in a standard risk group of adult B cell ALL patients who had achieved morphological remission following induction phase 1 therapy as part of the MRC UKALL12 protocol.Patients and Methodology. MRD tests were evaluated in fourty-seven patients with adult B cell ALL. They were negative for the t(9;22) or t(4;11) translocation and had received chemotherapy based treatment or auto stem cell transplant (A-SCT) only. Median age was 23 yrs (range 15.5–54.6 yrs); median WBC was 9.6 (range 1.1–163×109/l) with a predominance of common (28 pts) and pre B ALL (12 pts).All patients had at least one molecular marker which was tested by quantitative or semi-quantitative PCR with sensitivity ≥1E4. End points were either clinical relapse or disease free survival in complete remission with follow up ≥12 months. Time point for molecular evaluation were post induction phase 1, TP1 (median 0.9 mo; range: 0.6–1.8 mo) and post induction phase 2 (median 2.79 mo; range 1.8–3.5 mo).Results. Thirty three pts were tested at TP1 following morphological remission. Relapse free survival (RFS) analysis showed a statistically significant association between MRD positive tests (in 16 pts) and relapse, and MRD negative tests (in 17 pts) and CCR (p=0.03) (Figure 1, left diagram).Twenty five pts were analysed at TP2 following their early morphological remission. At this time point we observed the strongest association between MRD negative tests (in 13 pts) and CCR (in 10 pts) and MRD positive (12 pts) and relapse (in 11 pts) (p=0.01)(Figure 1, right diagram).Conclusions. Molecular monitoring of MRD shows that even among early morphological remitters a group of MRD positive patients can be identified that have poor overall outcome and may benefit from tailored therapies. Molecular assessment of residual disease should be used to stratify treatment in future adult ALL trials. [Display omitted]

Molecular Monitoring of Residual Disease (MRD) during Induction and Intensification Phases in Low Risk Adult B Cell ALL Treated According to the MRC UKALL12 Protocol.

Introduction and aims. Molecular monitoring of minimal residual disease (MRD) using Immunoglobulin (IG) and T cell receptor (TCR) targets has provided an independent and prognostically significant parameter of outcome in adult and childhood acute lymphoblastic leukaemia (ALL). The aim of our study was to evaluate the impact of molecular tests carried out during the first 20 to 24 weeks of chemotherapy for MRD in a standard risk group of adult B cell ALL patients.Patients and Methodology. We evaluated MRD tests in 63 patients with B cell ALL (37M/26F). Median age of our cohort was 24 yrs (range: 15.5–54.6 yrs) while median WBC was 9.9 (range: 1.1–553 × 109l). All were negative for the t(9;22) or t(4;11) translocation and received standard induction chemotherapy or auto stem cell transplant (A-SCT) only. All patients had at least one molecular marker which was tested by quantitative or semi-quantitative PCR with sensitivity ≥1E4 and all tests were carried out at time of morphological remission. End-points were relapse or continuous clinical remission (CCR) with follow up ≥12 months. Time point for molecular evaluation were post induction phase 1, TP1 (up to 1.8 mo), post induction phase 2 (1.8–3.5 mo), post intensification (3.5–5.7 mo). Median follow up was 92 months for patients in CCR (range 12–141 mo) and 15.5 months for patients who relapsed (range: 2.4–49 mo).Results. Thirty-five patients in complete morphological remission were tested at TP1. Relapse free survival (RFS) tests showed a statistically significant correlation (p=0.02) between MRD positive tests and relapse (n=19 pts) and MRD negative tests (n=16 pts) and relapse free survival. In addition, pts with resistant disease at TP1 (n=10; not included above) and MRD positive pts were indistinguishable as far as relapse FS and both faired extremely poorly (Figure A).Thirty patients were analysed at TP2. RFS confirmed the significant association between MRD positivity and poor outcome (n=16 pts) and MRD negativity and CCR (n=14 pts) (p=0.03) (Figure B).MRD data were also available for 43 patients at TP3. Association of MRD positive tests (n=15 pts) and MRD negative tests (n=28 pts) with poor and good outcome, respectively (p=0.0006) (Figure C) was strongest at this time point. Outcome correlated with level of MRD at all time points, with poorer outcome in patients with MRD >1E3.Conclusions. Molecular monitoring during induction and intensification for standard risk B cell ALL patients treated with UKALL12 protocol provides a prognostically significant parameter for the management of adult ALL in otherwise morphological remission and may in the future be used for patients' stratification. [Display omitted]

Does High Dose Cytosine Arabinoside Improves Disease Free Survival for Down Syndrome Acute Myelocytic Leukemia Patients?.

Acute myelocytic leukemia (AML) in Down Syndrome (DS) children is characterized by a young age of onset (< 2 years), a low white blood cell count and high frequency of Megakaryocytic leukemia. DS children with AML have higher disease free survival (DFS) rates as compared to non DS AML patients. Previous studies have suggested that intensification chemotherapy may not be necessary for the treatment of DS children with AML. The objective of this study was to clarify the effectiveness and toxicities of using high dose Cytosine Arabinoside (HD AraC) intensification in the treatment of DS AML. Clinical data for children (<14 years) with DS AML, diagnosed between September 2000 to May 2005, were retrieved from the hospital data base. Patients were divided into two groups; Group A patients received chemotherapy containing HD AraC, while Group B patients did not. A total of 15 patients were included, eight in Group A and seven in group B. The median age at diagnosis was 22 months (A=23 months, B=22 months). The two groups were matched regarding their clinical and laboratory parameters. There was no significant difference in DFS between groups A and B, 75% and 85% respectively (P = 0.82) at a mean observation period of 42.9 months for group A and 23.12 months for group B. The median time to relapse was 6 months for group A and 8 months for group B. The overall treatment related toxicity was higher in Group A patients but achieved only borderline significance (P = 0.06). However, when toxicity was assessed separately for induction and post induction phases of chemotherapy there were significantly more infectious events (17 v. 2; p=0.0006) in the post induction phase which includes HD AraC intensification in Group A. Even when only serious infections (bacteremia, fungal infection, sepsis) were included in the evaluation this difference persisted (7 v. 1; p=0.0339), with less toxicity for Group B patients. No such difference was noted between the two groups during induction chemotherapy. In conclusion the use of HD AraC in post-induction intensification phases for DS AML children does not improve DFS and is associated with more treatment related toxicity.

Effect of postinduction nutrient feed composition and use of lactose as inducer during production of thermostable xylanase in Escherichia coli glucose-limited fed-batch cultivations

Escherichia coli is a microorganism routinely used in the production of heterologous proteins. The overexpression of a xylanase (Xyn 10 A Delta NC), which originated from the thermophile Rhodothermus marinus cloned under the control of the strong T7/lac promoter in a defined medium (mAT) using a substrate-limited feed strategy, was however shown to impose a significant metabolic burden on host cells. This resulted in a decreased cell growth rate and ultimately also a decreased target protein production. The investigation hence centers on the effect of some selected nutrient feed additives (amino acid [Cys] or TCA-intermediates [citrate, succinate, malate]) used to relieve the metabolic burden imposed during the feeding and postinduction phases of these glucose-limited fed-batch cultivations. The use of either succinic acid or malic acid as feed-additives resulted in an increase in production of approximately 40% of the heterologous thermostable xylanase. Furthermore, use of lactose as an alternative inducer of the T7/lac promoter was also proven to be a suitable strategy that significantly prolonged the heterologous protein production phase as compared with induction using isopropyl beta-D-thiogalactopyranoside (IPTG).

The Kinetic of Reduction of Minimal Residual Disease Impacts on duration of Response snd Survival of Patients with Acute Myeloid Leukemia.

In acute myeloid leukemia (AML) patients achieving complete remission, the levels of minimal residual disease (MRD) as determined by flow cytometry have been shown to impact on remission duration and survival. However, some issues such as the most suitable source (BM or PB) or the most appropriate timing (early or delayed evaluation) for MRD determination are still a subject of debate. In our experience, we observed that MRD negativity, as defined by a number of bone marrow residual leukemic cells (BMRLC) <3.5x10−4, after consolidation cycle was associated with a significantly longer disease free survival (DFS) and overall survival (OS). Based on this, the present study was designed to analyze the kinetic of MRD reduction during the post-induction phase, and therefore to determine to what extent post-induction chemotherapy might impact on the outcome of patients with AML. Eighty-nine adult patients with AML were entered into the EORTC/GIMEMA protocols AML10/AML12 (age <61 yrs) or AML13/AML15 (age >61 yrs), all consisting in intensive induction and consolidation cycles, and allogeneic or autologous stem cell transplantation for patients aged < 61 years. Median age was 53 years (range 17–78), all FAB subtypes were represented with the exception of APL cases which were not included. Eighty-one of 89 patients maintained complete remission after consolidation (8/89 had early relapse after induction) and were suitable for the analysis. After consolidation cycle, 32 of 81 (39%) patients had <3.5x10−4 BMRLC and were considered MRD neg. The remaining 49 (61%), were MRD pos since the measured levels of MRD were ≥3.5x10−4 BMRLC. Among these MRD pos patients, in 16 the levels of MRD, although still above the value of 3.5x10−4 BMRLC, were significantly reduced as compared to the post-induction levels (median reduction 6x10−4 BMRLC, range 0–643). Therefore, these 16 patients were considered as having a “chemosensitive MRD” and in fact, within this MRD pos category, they had a superior duration of DFS and OS (P=.010 and .004, respectively) as compared to MRD pos patients with “chemoresistant MRD”, namely those not showing improvement in the level of MRD between the induction and consolidation course. Therefore, we identified 3 discrete categories of patients: 1) 32 patients MRD neg at the end of consolidation therapy (BMRLC <3.5x10−4); 2) 16 patients MRD pos at the end of consolidation therapy (BMRLC ≥3.5x10−4) but with “chemosensitive MRD”; 3) 33 patients MRD pos at the end of consolidation therapy (BMRLC ≥3.5x10−4) but with “chemoresistant MRD”. These 3 groups differed significantly in terms of relapse rate (84% vs. 75% vs. 28%, respectively) both in univariate and multivariate analysis (P<.001). Accordingly, DFS and OS (at 5-years, 66%, 50 % and 12%, respectively) (P<.001) duration also differed; the multivariate analysis confirmed the independent prognostic role of MRD status at the end of consolidation (P<.001). In conclusion, 1) at variance with previous reports emphasizing the prognostic value of an early flow-cytometric determination of MRD, we have found that a delayed (post-consolidation) MRD evaluation provides the best predictive information on patients outcome; 2) the quantitative determination of MRD at specific time-points may also allow the identification of MRD pos patients with variable prognosis (chemosensitive vs chemoresistant MRD).

Simultaneous determination of the content and activity of sterically hindered phenolic and amine stabilizers by means of an oxidative model reaction

Measurements of the reduced post-induction oxygen consumption rate in a model system comprising cumene initiated oxidation performed in the presence of the hindered amine stabilizer (HAS) Chimassorb 2020 offers the possibility of determining the state of the stabilizer type related to polymers. The rate of the post-induction oxidation depends linearly on the reciprocal square root of the concentration of the antioxidant over a sufficiently wide range thereby allowing reliable determinations to be made. The presence of the basic, sterically hindered phenolic stabilizer (Irganox 1010) usually used for polymer stabilization has no influence on the post-induction phase of cumene oxidation and conversely the HAS tested did not change the induction time and the rate constant induced by Irganox 1010. The rate constant for the addition of cumyl R radicals to Chimassorb 2020 was determined to be k (333 K) =(1.5±0.2)·10 7 M −1 s −1 . Thus the model reaction of initiated oxidation of cumene may be considered as a promising method for the simultaneous determination of phenolic and HAS-functionalities of stabilizers in polymers and their potential interactions.