International Collaborative Trial Research Articles

Contribution to development of remedies for COVID-19: focusing on Eritoran

Eritoran (E5564) is Eisai's in-house discovered and developed investigational Toll-Like Receptor 4 (TLR4) antagonist created with natural product organic synthesis technology. It is a structural analogue of Lipid A, which is an activator of endotoxins of bacteria. It has been previously observed to be safe in 14 clinical studies including a large Phase 3 randomized trial in severe sepsis. In order to evaluate therapeutic efficacy by eritoran, we are participating in the international network REMAP-CAP-COVID (Randomized, Embedded, Multi-factorial, Adaptive Platform-Community Acquired Pneumonia COVID) which aims for novel coronavirus medicine development through drug repurposing, and began an international collaborative clinical trial in October 2020 which is designated for confirmed novel coronavirus patients who are hospitalized and are in a progressing disease state. It is hoped that through suppressing the most upstream TLR4 activity which controls production of multiple cytokines by eritoran, the cytokine storm in patients can be suppressed and pneumonia can thus be prevented from becoming severe. On the other hand, E6011 is the only humanized anti-fractalkine (FKN) monoclonal antibody in the world created by KAN Research Institute. E6011 inhibits the tight binding of CD16-positive monocytes (a cell population that highly expresses the FKN receptor CX3CR1) to vascular endothelial cells, which are important for the local inflammatory response. This is expected to suppress the formation and exacerbation of vasculopathy in COVID-19.

Women are predisposed to early dose-limiting toxicities during adjuvant CAPOX for colorectal cancer.

Oxaliplatin-based adjuvant chemotherapy was demonstrated to be beneficial for stage III or high-risk stage II colorectal cancer (CRC). Moreover, a recent international collaborative trial suggested 3-months CAPOX as an alternative regimen for low-risk stage III colorectal cancer (CRC) patients. Thus, it is important to clarify the frequency and predictive markers of dose-limiting toxicities (DLTs) developed within the short-course CAPOX cycles. We investigated CRC patients who underwent radical surgery and adjuvant CAPOX therapy at our hospital between December 2010 and February 2021. Patients who received initially reduced doses of CAPOX and those who had early recurrence were excluded. We reviewed the age, sex, comorbidities, physical, laboratory and oncological data and other perioperative factors. The associations between these variables and early DLTs within four cycles of CAPOX were examined by multivariate analyses using logistic regression models. Among 168 patients (96 men, mean age: 58.3years), 120 (71%) developed early DLTs. Patients with early DLTs were predominantly women and sarcopenic and habitualalcohol consumers. On multivariate analyses, only the female sex was an independent predictive factor for early DLTs (odds ratio: 2.61, P=.027). Women were prone to early DLTs during adjuvant CAPOX in the current study. Doctors should be aware of the sex difference in the incidence of early DLTs, adjust the CAPOX dosage and provide supportive care for female CRC patients.

Contribution to Development of Remedies for COVID-19: Focusing on Eritoran

Eritoran (E5564) is Eisai's in-house discovered and developed investigational TLR4 (Toll-Like Receptor 4) antagonist created with natural product organic synthesis technology. It is a structural analogue of Lipid A, which is an activator of endotoxins of bacteria. It has been previously observed to be safe in 14 clinical studies including a large Phase 3 randomized trial in severe sepsis. We are participating in the international network REMAP-CAP-COVID (Randomized, Embedded, Multi-factorial, Adaptive Platform-Community Acquired Pneumonia COVID) which aims for novel coronavirus medicine development through drug repurposing, and began an international collaborative clinical trial in October 2020 which is designated for confirmed novel coronavirus patients who are hospitalized and are in a progressing disease state. It is hoped that through suppressing the most upstream TLR4 activity which controls production of multiple cytokines, the cytokine storm in patients can be suppressed and pneumonia can thus be prevented from becoming severe.

Stroke Avoidance for Children in República Dominicana (SACRED):a Prospective Trial to Reduce Stroke in Children with Sickle Cell Anemia

Introduction. The burden of sickle cell anemia (SCA) is high across the Caribbean, including the Dominican Republic where the morbidity of SCA is substantial due to limited resources. Approximately 10% of affected children will develop primary stroke, with serious medical and neurocognitive sequelae. In the US, transcranial Doppler (TCD) ultrasonography is an effective screening tool to identify children at risk for primary stroke, which then allows preventive therapy with either hydroxyurea or blood transfusions. To date, however, no routine TCD screening and treatment program for stroke prevention has been established in the Dominican Republic. We designed a prospective screening and treatment study (SACRED, NCT02769845) for prevention of primary stroke in children with SCA using open-label hydroxyurea at maximum tolerated dose (MTD). Methods. Children with SCA between age 3-15 years and receiving medical care at Robert Reid Cabral Children's Hospital in Santo Domingo, Dominican Republic were recruited over a 12-month period. Treatment depended on the baseline TCD examination: children with abnormal time-averaged mean velocities (≥200 cm/sec, high stroke risk) were recommended to receive monthly transfusions for primary stroke prevention, as per standard treatment preferences at the Robert Reid Hospital; those with conditional velocities (170-199 cm/sec, moderate stroke risk) were offered hydroxyurea study treatment unless they were currently receiving chronic transfusions for other clinical indications. Oral open-label hydroxyurea using 500mg capsules was prescribed at fixed-dose (20 mg/kg/day) for 6 months, followed by dose escalation to MTD. TCD examinations were repeated every 6 months, with the main study endpoints being changes in laboratory parameters and TCD velocities after 18 months of study treatment. Results. A total of 283 children were enrolled with the following baseline TCD velocities: 200 (70.7%) normal, 63 (22.3%) conditional, 11 (3.9%) abnormal, and 9 (3.1%) inadequate velocities. Among 50 children eligible for hydroxyurea study treatment, the average age was 6.8 ± 2.8 years and included 27 males/23 females with evidence of substantial SCA-related morbidity (88% with previous transfusions, 60% with >5 hospitalizations). A total of 48 children initiated hydroxyurea at 20.2 ± 1.2 mg/kg/day, which was maintained for 6 months followed by dose escalation, reaching MTD at 24.1 ± 6.0 mg/kg/day. Laboratory changes from baseline to Month 18 included significant increases in hemoglobin (7.5 ± 0.9 to 8.9 ± 1.3 g/dL, P<0.0001) and fetal hemoglobin (16.4 ± 7.4 to 30.9 ± 13.6%, P<0.0001). Serial TCD examinations revealed a significant decline over the treatment period, with the average TCD velocity decreasing from 180 ± 8 cm/sec at baseline to 159 ± 21 cm/sec at Month 18, P<0.0001. At Month 18, 68% of the originally conditional TCD examinations had reverted to a normal velocity, while only 4% had converted to abnormal velocity. No clinical strokes occurred in the hydroxyurea treatment cohort. Conclusion. Conditional TCD velocities are common (>20%) in the Dominican Republic, which documents an elevated stroke risk for children with SCA. The SACRED trial established a local TCD screening program and provided open-label hydroxyurea treatment for children with conditional TCD velocities. Hydroxyurea treatment at MTD led to improved hematological parameters, lower TCD velocities, and decreased stroke risk. SACRED represents an important international partnership and prospective collaborative research trial providing capacity building and valuable epidemiological data regarding TCD screening, stroke risk, and hydroxyurea effects for children with SCA. Hydroxyurea at MTD is a suitable treatment to reduce primary stroke risk for children with SCA living in the Dominican Republic or other low-resource settings. Disclosures Jeste: Jannsen: Employment. Ware:Novartis: Other: DSMB; Nova Laboratories: Membership on an entity's Board of Directors or advisory committees; CSL Behring: Membership on an entity's Board of Directors or advisory committees; Bristol Myers Squibb: Other: Research Drug Donation; Addmedica: Other: Research Drug Donation; Global Blood Therapeutics: Membership on an entity's Board of Directors or advisory committees; Agios: Membership on an entity's Board of Directors or advisory committees.

International collaborative ring trial of four gene-specific loop-mediated isothermal amplification assays in GMO analysis

We performed an international collaborative ring trial to validate gene-specific loop-mediated isothermal amplification (LAMP) assays targeting four exogenous transgenes commonly utilized in the development of genetically modified (GM) crops: bar, pat, cp4-epsps, and Cry1Ac, respectively. A total of 12 laboratories participated in the ring trial and returned their test results. Statistical analysis of the returned results showed high specificity and low limit of detection (LOD) of the four LAMP assays. The LOD for cp4-epsps, Cry1Ac, bar and pat genes assay was 5, 10, 20 and 20 haploid genome equivalents (HGE), respectively. Furthermore, good performance of the four assays was further demonstrated in the analysis of practical samples in which all participating laboratories returned expected and accurate test results. Altogether, the ring trial results confirmed that the four gene-specific LAMP assays can be utilized for quick screening of transgenic traits in routine laboratory analysis.

679 Bismuth Classification of Hilar Cholangiocarcinomas Accurately Predicts the Clinical Outcomes and Survival Rates of Patients that Undergo Unilateral Versus Bilateral Endoscopic Palliative Stenting: Results of the Multicenter, International Collaborative Trial

679 Bismuth Classification of Hilar Cholangiocarcinomas Accurately Predicts the Clinical Outcomes and Survival Rates of Patients that Undergo Unilateral Versus Bilateral Endoscopic Palliative Stenting: Results of the Multicenter, International Collaborative Trial

Su1297 A Comparative Study Evaluating Bi-Flanged Fully Covered Self-Expanding Metal Stents and Lumen-Apposing Metal Stents for the Endoscopic Drainage of Pancreatic Walled-Off Necrosis: Results of the Multicenter, International Collaborative Trial

Su1297 A Comparative Study Evaluating Bi-Flanged Fully Covered Self-Expanding Metal Stents and Lumen-Apposing Metal Stents for the Endoscopic Drainage of Pancreatic Walled-Off Necrosis: Results of the Multicenter, International Collaborative Trial

Interlaboratory validation of an LC-MS/MS method for the determination of melamine and cyanuric acid in animal feed

ABSTRACTMelamine and cyanuric acid have been mixed illegally into food and feed to increase the nitrogen content, which results in deceptively high protein contents. As a consequence, a maximum level for melamine of 2.5 mg kg–1 feed was established by the European Union under Directive 2002/32/EC. The Technical Committee (TC) 327 of the European Committee for Standardisation (CEN) commissioned the standardisation of a method for the analysis of melamine and cyanuric acid in animal feed. One main task in the standardisation process is the performance of a full international collaborative trial, which is described in this paper. After performing a pre-trial study, in the main study eight different feed samples with different concentration levels of melamine and/or cyanuric acid were distributed as double-blind samples to 13 participants. The minimum criterion of eight laboratories submitting results per sample is fulfilled for melamine but only partly for cyanuric acid. The evaluation showed for both analytes a Horwitz ratio (HorRat) well below 2, and meets the requirements stated in the appropriate international protocols. The results demonstrated that the method seems to be suitable for the analysis of melamine and cyanuric acid in animal feed.

Quantitation of Compounds in Wine Using (1)H NMR Spectroscopy: Description of the Method and Collaborative Study.

To examine whether NMR analysis is a suitable method for the quantitative determination of wine components, an international collaborative trial was organized to evaluate the method according to the international regulations and guidelines of the German Institute for Standardization/International Organization for Standardization, AOAC INTERNATIONAL, the International Union of Pure and Applied Chemistry, and the International Organization of Vine and Wine. Sugars such as glucose; acids such as malic, acetic, fumaric, and shikimic acids (the latter two as minor components); and sorbic acid, a preservative, were selected for the exemplary quantitative determination of substances in wine. Selection criteria for the examination of sample material included different NMR spectral signal types (singlet and multiplet), as well as the suitability of the proposed substances for manual integration at different levels of challenge (e.g., interference as a result of the necessary suppression of a water signal or the coverage of different typical wine concentration ranges for a selection of major components, minor components, and additives). To show that this method can be universally applied, NMR measurement and the method of evaluation were not strictly elucidated. Fifteen international laboratories participated in the collaborative trial and determined six parameters in 10 samples. The values, in particular the reproducibility SD (SR), were compared with the expected Horwitz SD (SH) by forming the quotient SR/SH (i.e., the HorRat value). The resulting HorRat values of most parameters were predominantly between 0.6 and 1.5, and thus of an acceptable range.

Efficacy and Safety of Radiofrequency Ablation Versus Observation for Nonfunctioning Benign Thyroid Nodules: A Randomized Controlled International Collaborative Trial

Percutaneous radiofrequency thermal ablation (RFA) has been reported as an effective tool for the management of benign thyroid nodules (BTN). However, large, randomized controlled trials (RCTs) are lacking. The aims of this study were to assess the volume reduction of BTN after a single RFA performed using the moving-shot technique and to compare the volume reduction obtained in patients treated in two centers with different experience of the moving-shot technique. This study was an international prospective RCT. It was carried out at the Mauriziano Hospital (Turin, Italy) and the Asan Medical Center (Seoul, Korea). Eighty patients harboring solid, compressive, nonfunctioning BTN (volume 10-20 mL) were enrolled. Twenty patients in each country were treated by RFA using a 18-Gauge internally cooled electrode (group A); 20 nontreated patients in each country were followed as controls (group B). At six months, BTN volume significantly decreased in group A (15.1±3.1 mL vs. 4.2±2.7 mL; p<0.0001), whereas it remained unchanged in group B (14.4±3.3 mL vs. 15.2±3.5 mL). The baseline volume was larger in the Italian series (16.4±2.5 mL vs. 13.9±3.3 mL, p=0.009). However, at six months, there was no significant difference between the Korean group and the Italian group (3.7±2.9 mL vs. 5.5±2.2 mL). Both cosmetic and compressive symptoms significantly improved (3.6±0.5 vs. 1.7±0.4 and 3.6±1.9 vs. 0.4±0.7, respectively; p<0.001). No side effects occurred. RFA was effective in reducing the volume of BTN. The outcome was similar in centers with different experience in the moving-shot technique.

Collaborative trial validation of cry1Ab/Ac and Pubi-cry TaqMan-based real-time PCR assays for detection of DNA derived from genetically modified Bt plant products

Presence of genetic modifications in rice products originating from China and imported to the European Union market is detected since 2006. Neither these products from China nor any other genetically modified rice lines are approved as food or feed in the EU. The transgenic rice varieties identified contain genetic elements and constructs coding for insect-resistance genes from Bacillus thuringiensis (Bt) coding for insecticidal crystal (cry) proteins. In particular, DNA sequences coding for codon-optimised or fused cry1Ab/c genes and constructs driven by the maize ubiquitin promoter (P-ubiZM1) were identified. For improved screening and identification of genetic modifications present in Asian rice products, two TaqMan-based real-time PCR assays targeting codon-optimised cry1Ab/Ac and the Pubi-cry construct have been developed. These assays have been validated in an international collaborative trial with 17 participants from 10 countries. Based on a new mathematical–statistical model and an adjusted experimental set-up of the collaborative trial, a close examination of the limit of detection (LOD95%) and the probability of detection of the qualitative PCR assays was conducted. The evaluation of the method performance characteristics and results of the collaborative trial validation are presented.

Editor’s Spotlight/Take 5: What Are the Levels of Evidence on Which We Base Decisions For Surgical Management of Lower Extremity Bone Tumors?

Editor’s Spotlight/Take 5: What Are the Levels of Evidence on Which We Base Decisions For Surgical Management of Lower Extremity Bone Tumors?

A phase I/II study of the vascular disrupting agent BNC105P in combination with gemcitabine-carboplatin in partially platinum-sensitive ovarian cancer patients in first or second relapse: An international collaborative group trial of ANZGOG and HOG.

TPS5612 Background: BNC105P is a tubulin polymerization inhibitor and a vascular disrupting agent (VDA). In vivo exposure to BNC105P leads to selective damage of tumor vasculature in both primary and metastatic lesions, causing disruption of blood flow to tumors, hypoxia, and associated tumor necrosis. BNC105P also has a direct anti-proliferative action on cancer cells, including ovarian cancer cell lines. Pre-clinical data has demonstrated synergistic activity of BNC105P when combined with platinum or with gemcitabine, supporting the proposed study design. This study will determine the safety and efficacy of BNC105P in ovarian cancer when used in combination with gemcitabine-carboplatin. The target population is women with ovarian or primary peritoneal cancers who progressed 4 to 9 months after first-line platinum based chemotherapy, or 4 to 12 months after second line platinum based chemotherapy. Methods: A single arm phase I will be used to determine the phase II dose for the triplet combination (3-6 subjects per dose level, maximum of 24 subjects). Four dose levels of BNC105P (12-16 mg/m2) and gemcitabine (800-1000 mg/m2) will be assessed. The dose of carboplatin will be set at AUC 4. Enrolment to cohort 2 started in January 2013. The phase II component will consist of a 2-arm, randomized (1:1) study of BNC105P, gemcitabine and carboplatin versus gemcitabine and carboplatin alone. The primary endpoint for the phase II trial is objective response rate (ORR, according to RECIST 1.1 and/or GCIG CA125 criteria. An ORR of 40% or more with the experimental regimen would be considered worthy of further investigation, assuming an ORR of 20% with the control regimen. 110 phase II participants are planned (N = 55/arm). Treatment allocation will be balanced using minimization for the study site, target lesions according to RECIST (present vs. absent), progression free interval from last platinum based chemotherapy regimen (&lt;6 months vs 6 months or more), and first relapse vs. second relapse. Biomarker (tissue and blood-borne) sampling and PK analysis will also be undertaken. Clinical trial information: NCT01624493.

Abstract P6-07-05: Prognosis of 368 women with primary breast cancer during pregnancy: results from an international collaborative trial

Abstract Objective: We aimed to add to the limited outcome data on breast cancer during pregnancy (BCP), and compared disease-free survival (DFS), and overall survival (OS) between patients with BCP and breast cancer not diagnosed during pregnancy, adjusted for known prognostic factors. Methods: We analyzed a pro- and retrospectively compiled multi-centric registry of BCP patients (www.cancerinpregnancy.org and GBG/BIG 0203). Outcome was compared to a control group of patients who did not have associated pregnancies. The main analysis is a Cox proportional hazards regression of disease free survival (DFS) and overall survival (OS) on exposure (pregnant or not). The analysis was weighted by propensity score matching to correct for any bias that may have been associated with differences in baseline characteristics, with the following set of covariates: age, stage, grade, hormone receptor status, HER2 status, histology, type of chemotherapy and trastuzumab. Sensitivity analysis was also performed on age only, and also on chemotherapy only. Results: From April 2003-December 2011, 447 women with BCP were registered, 368 women with BCP were eligible for this analysis. The control group consisited of 2739 women (ratio 1:7.44). Median age was 33 years (interquartile range 30–36) for the cases and 41 years (interquartile range 37–43) for the controls. Median follow-up was 57 months. There were no statistically significant differences noted for DFS and OS studied after propensity-score matching, and sensitivity analysis. Conclusion: Patients diagnosed with breast cancer during pregnancy do not have a worse prognosis compared to patients who are not pregnant after matching for prognostic factors and treatment. This information is important when BCP patients are counseled regarding prognosis and adds to the possibility of treatment during pregnancy. Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr P6-07-05.

International Pig‐a gene mutation assay trial (Stage III): Results with N‐methyl‐N‐nitrosourea

N-methyl-N-nitrosourea (MNU) was evaluated in the in vivo Pig-a mutation assay as part of an International Collaborative Trial to investigate laboratory reproducibility, 28-day study integration, and comparative analysis with micronucleus (MN), comet, and clinical pathology endpoints. Male Sprague Dawley rats were treated for 28 days with doses of 0, 2.5, 5, and 10 mg MNU/kg/day in two independent laboratories, GlaxoSmithKline (GSK) and Bristol Myers Squibb (BMS). Additional studies investigated the low-dose region (<2.5 mg/kg/day). Reticulocytes were evaluated for Pig-a phenotypic mutation, CD59-negative reticulocytes/erythrocytes (RETs(CD592-)/ RBCs(CD592-)) on Days 1, 4, 15, 29, 43, and 57, and for micronucleated reticulocytes (MN-RETs) on Days 4 and 29. Comet analysis was conducted for liver and whole blood, and hematology and clinical chemistry was investigated. Dose-dependent increases in the frequency of RETs(CD592-) and RBCs(CD592-) were observed by Day 15 or 29, respectively. Dose-dependent increases were observed in %MN-RET on Days 4 and 29, and in mean %tail intensity in liver and in blood. Hematology/clinical chemistry data demonstrated bone marrow toxicity. Data comparison between GSK and BMS indicated a high degree of concordance with the Pig-a mutation assay results, consistent with previous observations with MNU and N-ethyl-N-nitrosourea. These data confirm that complementary genotoxicity endpoints can be effectively incorporated into routine toxicology studies, a strategy that can provide information on gene mutation, chromosome damage, and DNA strand breaks in a single repeat dose rodent study. Collectively, this would reduce animal usage while providing valuable genetic toxicity information within the context of other toxicological endpoints.

Interlaboratory Pig‐a gene mutation assay trial: Studies of 1,3‐propane sultone with immunomagnetic enrichment of mutant erythrocytes

An international collaborative trial was established to systematically investigate the merits and limitations of a rat in vivo Pig-a gene mutation assay. The product of this gene is essential for anchoring CD59 to the plasma membrane, and mutations in this gene are identified by flow cytometric quantification of circulating erythrocytes without cell surface CD59 expression. Initial interlaboratory data from rats treated with several potent mutagens have been informative, but the time required for those flow cytometric analyses (∼20 min per sample) limited the number of cells that could be interrogated for the mutant phenotype. Thus, it was desirable to establish a new higher throughput scoring approach before expanding the trial to include weak mutagens or nongenotoxicants. An immunomagnetic column separation method that dramatically increases analysis rates was therefore developed (Dertinger et al. [2011]: Mutat Res 721:163-170). To evaluate this new method for use in the international collaborative trial, studies were conducted to determine the mutagenic response of male Sprague Dawley rats treated for 3 or 28 consecutive days with several doses of 1,3-propane sultone (1,3-PS). Pig-a mutant frequencies were measured over a period of several weeks and were supplemented with another indicator of genetic toxicity, peripheral blood micronucleated reticulocyte (MN-RET) counts. 1,3-PS was found to increase Pig-a mutation and MN-RET frequencies in both 3- and 28-day study designs. While the greatest induction of MN-RETs was observed in the 3-day study, the highest Pig-a responses were found with 28-days of treatment. Pig-a measurements were acquired in approximately one-third the time required in the original method, while the number of erythrocyte and reticulocyte equivalents analyzed per sample were increased by factors of 100 and 10, respectively. The data strongly support the value of using the immunomagnetic separation technique for enumerating Pig-a mutation frequencies. These results also demonstrate that the ongoing international trial will benefit from the inclusion of studies that are based on both acute and protracted repeat dosing schedules in conjunction with the acquisition of longitudinal data, at least until more data have been accumulated.

Renaissance of contact x-ray therapy for treating rectal cancer

Contact x-ray therapy (CXRT) with 50 kV has proven to be an efficient radiation therapy technique to achieve local control and rectal preservation for early rectal adenocarcinoma. Despite these results, CXRT has not been used due to the shortage of the no longer manufactured Philips RT 50™ unit. Recently, a new CXRT machine (Papillon 50™) became available on the market. This machine delivers a beam of 50 kV with a dose rate close to 15 Gy/min and has a percentage depth dose of 50% at 6–7 mm. The applicator size varies from 2–3 cm in diameter. Due to the original design of the main tube, treatment delivery is quick and more comfortable for the patients. An online viewing system incorporated in the tube allows a good visualization of the tumor with improved accuracy of radiation delivery. An international collaborative trial (Contact Endoscopic Microsurgery [CONTEM]) was set up to accrue approximately 300 cases of rectal adenocarcinoma staged T1, T2 or early T3 tumors in the UK, France, Denmark and Sweden. This trial should confirm the role of CXRT in curative treatment with organ preservation for early rectal cancers.

Development and validation of stable reference materials for food microbiology using Bacillus cereus and Clostridium perfringens spores

To develop a new type of microbiological Reference Materials (RMs), displaying long-term stability at room temperature. The purpose was to produce and validate two batches of RMs for the enumeration of Bacillus cereus and Clostridium perfringens. The RMs were based on spores of B. cereus and Cl. perfringens, adsorbed on calcium carbonate pellets. Two batches of 1000 units were manufactured and validated in compliance with ISO guide 35. After verification of their homogeneity, the stability of the 'RM-B. cereus' and 'RM-Cl. perfringens' batches was proven during at least 36 and 9 months, respectively, at room temperature. The validation study was completed by international collaborative trial involving 12 laboratories, allowing the validation of the assigned values. The methodology developed in this work enabled to produce easy-to-handle and cost-effective RMs, displaying an unprecedented stability at room temperature, a good homogeneity and a precise and validated assigned value. This study revealed new paths for the development of stable microbiological RMs. Overcoming the intrinsic instability of the living cells makes it possible to produce valuable tools for the quality assurance of microbiology laboratories.

Determination of ester‐bound 3‐chloro‐1,2‐propanediol and glycidol in fats and oils – a collaborative study

AbstractIn 2009, the Joint Committee of DIN and DGF for the Analysis of Fats, Oils, Fatty Products, Related Products and Raw Materials (GA FETT) published the German Standard Method C‐III 18 (09) – Ester‐bound 3‐chloropropane‐1,2‐diol (3‐MCPD‐Esters) and 3‐MCPD forming substances – Determination in fats and oils by GC‐MS. This method was validated in a collaborative trial organised by the Federal Institute for Risk Assessment (BfR). Following further work on bound 2,3‐epoxy‐1‐propanol (glycidol) as a possible precursor for 3‐MCPD‐Esters, the German Standard method was modified in 2009 to determine ester‐bound 3‐chloro‐1,2‐propandiol and 2,3‐epoxy‐1‐propanol. The modified DGF standard method, together with a similar method, developed by SGS Hamburg (Kuhlmann method), was validated in an international collaborative study. The results of this collaborative study are reported for vegetable fats and oils, using GC with mass spectrometric detection and two different deuterated internal standards. Altogether, 17 laboratories from 5 countries (D – E – F – HU – USA) expressed interest in participation. The test was carried out according to ISO 5725:1994 – Accuracy (trueness and precision) of measurement methods and results and the Guidelines for Collaborative Study Procedures To Validate Characteristics of a Method of Analysis (AOCS 2002). The results are reported.Practical application: For the quantitative determination of 3‐MCPD‐ and glycidyl‐esters in refined fats and oils standardised methods are not yet available. Two similar methods for the quantification of the sum of ester‐bound 3‐MCPD and glycidol and for ester‐bound 3‐MCPD alone were tested in an international collaborative trial.

The Paediatric LMB Protocol Has High Curative Potential in Adults with Burkitt Lymphoma and High Proliferation DLBCL.

Abstract 3702Poster Board III-638 BackgroundThe optimum treatment for adults with aggressive, high proliferation rate B-cell NHL remains uncertain. A proportion are classified as Burkitt Lymphoma (BL), based on typical morphology, immunophenotype and the presence of a MYC rearrangement (MYC+); the remainder as diffuse large B-cell lymphoma (DLBCL) or intermediate BL/DLBCL. Recent gene-expression studies report up to 30% of cases classified as DLBCL by morphology have the genetic signature of BL. BL is most commonly treated in the UK using CODOX-M/IVAC. Non-Burkitt, high-proliferation DLBCL is most commonly treated with R-CHOP. A recent UK study of CODOX-M/IVAC for B-NHL with >95% proliferation achieved 2yr EFS for BL 64% (34/53), DLBCL 54% (31/57). A recent large international collaborative trial using the risk-adapted LMB protocol for both BL and DLBCL in children and adolescents to age 21 achieved 4yr EFS of 80% for patients with B-ALL or CNS disease, and >85% for all others. The EFS for BL and DLBCL were identical. In recent years we have adopted the LMB strategy for treating younger adults diagnosed as BL, Burkitt-like lymphoma or mature B-ALL. We report the outcome of 22 adults treated in a single institution. MethodsCase histology was reviewed and re-classified as BL if characteristic morphology, phenotype (CD20+ CD10+ Bcl-6+ Bcl-2–) and, where tissue available, presence of rearranged MYC by FISH. DLBCL cases had high proliferation (≥95%) but did not fulfill criteria for BL. Intermediate BL/DLBCL included DLBCL with MYC+. There were no BCL2+/ MYC+ “double hit” lymphomas. B-ALLs had a purely marrow presentation with vacuolated blasts and mature B phenotype. All patients had staging CT and PET, and CNS MRI where brain/spinal involvement suspected. Treatment was based on the LMB 86/89 protocol with 3 levels of intensity as published (Patte C et al. Blood 2001 97:3370). All received low-dose vincristine, cyclophosphamide, prednisolone induction 7 days prior to 2, 5 or 8 intensive chemotherapy blocks as dictated by stage, marrow and/or CNS involvement. The interval between the start of the first 2 intensive blocks was kept strictly to 21 days. HIV positive cases were treated concomitantly with HAART. ResultsTwenty two consecutive cases have been treated, 11 BL, 6 DLBCL, 2 intermediate BL/DLBCL, 3 B-ALL. Eleven cases were HIV positive, 2 others followed renal transplant. Median age 37 years (range 15-49) plus 1 older patient of 62 yrs. Lymphoma case IPI scores I (n=6), II (4), III (4), IV (5). Pre-treatment PET scanning increased clinical stage in 10 cases and escalated planned treatment from schedule A to B in 3 cases. One patient received LMB schedule A (resected stage I), 7 schedule C for B-ALL (3) BL with marrow blasts >70% (1) or CNS disease (3), all others (14) received schedule B. High dose methotrexate (MTX) dose was only reduced in the two renal transplant patients to limit toxicity to the transplanted kidney, as guided by MTX levels. Neutropenia was mostly short-lived and G-CSF used infrequently to prevent treatment delay. One third of patients experienced significant sepsis. Two HIV patients experienced complications of CMV reactivation. All HIV and renal transplant patients experienced significant post-MTX mucositis, severe in 5. In non-immunosuppressed patients mucositis was mild. All patients had >50% response to the 3 drug induction. All 22 patients achieved CR defined by PET ± CT. There were no treatment related deaths. Three relapsed from CR within 3 months of completing chemotherapy: 1 had B-ALL without MYC+, the other 2 were HIV+, MYC+ BL. All 3 died from disease progression during salvage therapy. The 62 yr old died of HIV in CR 2yrs post Rx. The remaining 18 patients are alive and in CR at median 40 (8-177) months from diagnosis (Relapse rate 14%; PFS 82%). ConclusionsThe risk-adapted LMB protocol is safe, with manageable toxicity in adults <50 years. Outcomes in this series compare favourably with CODOX-M/IVAC. As in children, our experience suggests the LMB protocol may be equally effective for BL and high proliferation DLBCL in adults, thus circumventing the need for diagnostic separation of two diseases whose differentiation based on histology alone is problematic. These data suggest the LMB protocol warrants testing by randomised trial in younger adults with high proliferation DLBCL and BL. Disclosures:No relevant conflicts of interest to declare.